review of literatureshodhganga.inflibnet.ac.in/bitstream/10603/6809/6/06... · 2015-12-04 ·...
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CHAPTER II
REVIEW OF LITERATURE
The available published information and studies that are most pertinent to the subject of
this study were reviewed and presented under the following headings:
2.1. History of aflatoxin
2.2. Chemistry of aflatoxin
2.3. Incidence of aflatoxin
2.4. Metabolism of aflatoxin
2.4.1. Absorption, Distribution, Biotransformation and Excretion
2.4.2. Toxicity and mode of action
2.5. Aflatoxicosis
2.5.1. Aflatoxicosis in commercial layers
2.5.2. Aflatoxicosis in breeders
2.5.2.1. Aflatoxin residue in eggs
2.5.3. Aflatoxicosis in broilers
2.5.3.1. Growth, feed consumption, mortality and organ weights
2.5.3.2. Feed conversion ratio
2.5.3.3. Gross lesions
2.5.3.4. Plasma proteins
2.5.3.5. Serum enzyme activity
2.5.3.6. Immune system and antibody response
2.6. Counteraction of aflatoxicosis
2.6.1. Physical methods
2.6.1.1. Zeolites
2.6.1.2. Hydrated sodium calcium, aluminosilicate (HSCAS)
2.6.1.3. Activated carbon
2.6.1.4. Bentonite
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2.6.1.4.1. Physical and chemical properties
2.6.1.4.2. Surface chemistry
2.6.1.4.3. Uses
2.6.1.5. Other adsorbent compounds
2.6.2. Herbal methods
2.6.2.1. Antioxidant agents
2.6.2.2. Vitamin and related substances
2.6.2.3. Herbal compounds
2.6.2.4. Spirulina platensis
2.6.3. Use of enzymes
2.6.4. Nutritional manipulations
2.6.5. Biological methods
2.6.5.1. Bacterial degradation
2.6.5.2. Protozoan degradation
2.6.5.3. Fungal degradation
2.6.5.4. Degradation by yeast
2.6.5.5. Counteraction of aflatoxin by Mannanoligosaccharide
2.6.5.5.1. Oligosaccharide
2.6.5.5.2. Mannanoligosaccharides derived from cell wall
2.6.5.5.3. Mode of action of Mannanoligosaccharide
2.6.5.5.3.1. Blocking of colonization by pathogens
2.6.5.5.3.2. Stimulation of immune response
2.6.5.5.3.3. Mycotoxin adsorption
2.6.5.5.3.3.1. Glucomannan - Extract Binder
2.1. History of aflatoxin
Aflatoxin is the most important fungal toxin, which contaminates feeds and feed stuffs
causing aflatoxicosis. During the spring and summer of 1960, in Southern and Eastern
England, numerous outbreaks of a disease of an unusual nature occurred in turkey poults,
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characterized by acute hepatic necrosis with bile hyperplasia, loss of appetite, lethargy, wing
weakness, unusual posture of the heads and neck at the time of death. The condition was
named as “turkey X disease” and the cause was explored and later designated as aflatoxicosis
(Asplin and Carnaghan, 1961). Later on, a toxic factor in Brazilian groundnut meal was
reported to be responsible for the disease and the toxins were identified as metabolites of some
strains of Aspergillus flavus. The toxin factor was given the name “Aflatoxin” (Carnaghan and
Sargeant, 1961).
World wide occurrence of aflatoxins in food and feeds is well documented. Majority of
the mentioned grains contained aflatoxins above 20µg/kg which is the regulatory limit in feed
all over the world. Although the incidence of aflatoxicosis has been recognized in ducklings as
early as 1963, in India an outbreak of aflatoxicosis in poultry was recorded only in 1968
(Gopal et al., 1968).
It has been proved that chicks are more resistant to aflatoxins compared to turkey poults
and ducklings. The main effect of aflatoxicosis in chicken is retardation of growth. Ducklings
are most susceptible and within three to four days after feeding the toxic meal, extensive
proliferation of bile duct epithelial cell was clearly visible (Durate and Smith, 2005).
2.2. Chemistry of Aflatoxin
Aflatoxin (AF) consists of heterocyclic metabolites produced by toxigenic species of
fungi Penicillium puberulum (Smith and Hamilton, 1970), Aspergillus flavus, Aspergillus
parasiticus (Giambrone et al., 1985) and A. nomius (Ellis et al., 1991). Other fungi involved in
AF production are A. bombycis, A. ochraceoroseus and A. pseudotamari (Mishra and Das,
2003). However, AF is predominantly produced by Aspergillus flavus and Aspergillus
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parasiticus during their growth on feed and food as a common contaminant. A. flavus produces
mainly AFB1 and B2 while A. parasiticus produces AFB1, B2, G1 and G2 (Davis and Diener,
1983).
There are 18 different aflatoxins which are divided into two chemical groups; the
difurocoumarocyclopentenone series (AFB1, B2, B2A, M1, M2, M2A and aflatoxicol and the
difurocoumarolactone series (AFG1 and G2). Chemically, aflatoxins are difurocoumarin
derivatives (Buchi and Rae, 1969) and structurally consist of a bifuran ring fused to coumarin
nucleus with a pentenone ring in B and M aflatoxins on a six-member lactone ring in G
aflatoxins. The most important ones are AFB1, B2, G1 and G2.
The aflatoxins Bs and Gs are separated by the color of fluroscence under long waves
and ultraviolet illumination i.e. B-blue and G-green. Melting points of AF are 269, 238, 245
and 239°C for AFB1, B2, G1 and G2, respectively. Molecular formulae for AFB1, B2, G1 and G2,
are C17H12O6, C17H14O6, C17H12O7 and C17H14O7, respectively.
The synthesis of AF by Aspergillus species was favorable at 12 to 41°C and optimum
production occurred between 25 and 35°C (Lillehoj, 1983). There was an increase in the
production of AFB1, B2, G1 and G2 in feed at temperature above 27°C, humidity levels above
62 per cent and moisture levels above 14 per cent (Royes and Yanong, 2002).
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2.3. Incidence of aflatoxin
It is imperative that food contaminated with AF is considered unsafe for human and
animal health. Aflatoxins occur over a wide variety of substrates of practical importance to
poultry feeding (maize, groundnut/meal, cottonseed meal, sunflower extractions, rice, soya
bean meal and compounded feeds.) Because of increasing awareness of the risk of AF
contamination of foods and feeds, this has opened a new vista to conduct survey of feed stuffs
which are commonly contaminated with AF. The details of the survey are presented in Table
2.1.
Results of the contamination monitoring program for mycotoxins from 1976 to 1983
showed that, much of the monitored grain contained AF above 20.00 to 50.00ppb, higher than
the regulatory limits in feeds of most countries (Jelinek et al., 1989).
2.4. Metabolism of aflatoxin
Metabolism or biotransformation plays an important role in the biological activity and
disposition of aflatoxins. The details reviewed are presented here under:
2.4.1. Absorption, Distribution, Biotransformation and Excretion
The main sites for absorption of Aflatoxins are gastrointestinal tract (GIT), lungs and
skin. Aflatoxins are readily absorbed from the site of exposure into the blood stream, as they
are highly lipo soluble (Sawhney et al., 1973). After absorption from GIT, AF enters into blood
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and accumulates in most of the soft tissues and body fat depots, the accumulation being
greatest in liver and kidney (Harland and Cardeilhac, 1975).
Table 2.1 : The results of surveys conducted by various investigators on natural
occurrence of aflatoxins in various feeds and feed stuffs
Author and year Ingredient Level (ppb)
Shetty et al. (1987) Mixed poultry feed 30-1610
Jelinek et al. (1989) Corn and corn products Peanuts
0.1-1970 0.2-5000
Devegowda et al. (1990) Groundnut
Maize Bajra
48-900 32-1000
12-15
Hegazy et al. (1991) Poultry feed 1-2000
Devegowda and Arvind (1993) Maize
Ground nut cake Others
25-1002 45-1500
10-80
Jindal et al. (1993) Poultry feeds > 300 30-160
Dhavan and Choudary (1995) Feed ingredients and mixed feeds High concentrations
Sala and Ueno (1997) Maize 20-100
Chandrasekharan (2000) Maize 21-100
101-500 500
Pandey et al. (2001)
Maize Wheat
Groundnut extraction
948 285 225
Chandrasekharan et al. (2002) Different feed samples 0 - 50
Wang et al. (2003) Different feed samples 8.27
Manafi, (2006) Poultry feeds 500
In the case of aflatoxins, biotransformation plays a major role in the disposition and
toxicological activity. Bioactivation has been demonstrated as a prerequisite for most of the
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toxic and carcinogenic effects of Aflatoxins. AFB1 requires biotransformation to AF 2-3
epoxide before exerting its biochemical effects. The basic enzymatic biotransformation phase I
reactions (oxidation, reduction and hydrolysis) and its metabolites are conjugated (Phase II
reaction) with endogenous substances in order to facilitate excretion. AFB1 is biotransformed
by cytochrome P-450 into several water-soluble metabolites including AFM1 and is excreted
rapidly through urine and faeces (Sawhney et al., 1973).
2.4.2. Toxicity and mode of action
Aflatoxin B1 is found to be highly toxic (6.1mg/kg body weight) to chicken as
compared to other Aflatoxins. Chronic aflatoxicosis resulting from regular low level dietary
intake of AF caused reduced weight gain, decrease in feed intake and poor feed efficiency. The
important biochemical effects of AFB1 are inhibition of DNA replication and RNA synthesis
(Kichou and Walser, 1994). Hsieh (1985) reported inhibition of elongation and/or termination
of the translational process of protein synthesis, interference in successive steps in
mitochondrial respiratory chain, alteration in immune response and exert carcinogenic,
teratogenic and mutagenic effects by reacting with nucleophillic sites in macromolecular
components. Further, it was stated that AF is accumulated in liver and the high content of
microsomal cytochrome P-450 enzymes of hepatic cells favors the formation of DNA - AF
adducts. Hence, liver is the major target organ for the AF toxicity. Among avian species, the
most susceptible are ducks and turkeys followed by pheasants, chickens and quails.
2.5. Aflatoxicosis
Aflatoxicosis caused by consumption of aflatoxins represents one of the most serious
diseases to man, as well as poultry, livestock and other animals.
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Aflatoxicosis in poultry is characterized by hemorrhages, anorexia, mortality, decreased
feed efficiency and production, pathological changes in the liver, kidney and bile duct. The
economic loss in the poultry industry due to aflatoxicosis is estimated to run upto millions of
dollars (Raju et al., 2005).
2.5.1. Aflatoxicosis in commercial layers
The most prominent manifestations of experimental aflatoxicosis in layers are reduced
egg production and egg weight, increased liver fat and alterations in some serum biochemical
parameters.
Sims et al. (1970) fed ad libitum AF-contaminated diet having levels of 2.00 to
8.00ppm AFB1 for 17 days and observed a significant reduction in egg production. Egg weight
was not affected and also they could not detect any fluorescent metabolites in the eggs or liver
of hens fed dietary AF.
Hamilton and Garlich (1971) fed the Single Comb White Leghorn hens with 1.25-
200ppm dietary AF for three weeks and reported a dose related decrease in egg production and
egg size, but shell thickness was not affected. The lipid content of liver was significantly
increased in AF fed hens (5.00ppm) when compared with the control group.
Garlich et al. (1973) reported that the White Leghorn hens receiving 20.00ppm of AF
in their diet for seven days did not adversely affect egg production but plasma calcium, protein,
cholesterol and triglycerides were all decreased. In this study, delayed adverse effect of AF on
egg production was observed. Once the hens were returned to a control diet for recovery, egg
production began to decline significantly from the first day of the recovery period. Egg
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production reached to a minimum of 35 per cent, seven days later and then returned to the level
of the control group, 19 days after the withdrawal of the contaminated diet. This delayed effect
on egg production emphasizes the severe epidemiological problem of mycotoxins. Under field
conditions, the feed causing the problem can be totally consumed before its adverse effects are
noticed to undertake any therapeutic measure to solve the problem.
Huff et al. (1975) investigated the effect of graded levels of dietary AF up to 10.00ppm
on layers. After four weeks, liver size and liver lipid content were increased, while egg
production and egg size were decreased. Dry weight and lipid content of the yolk were not
affected but yolk and plasma carotenoid concentrations were elevated.
McDaniel et al., (1979) reported that feeding of 200ppb AF in the diets did not
significantly alter shell thickness of eggs obtained from layers. They concluded a trend with the
known phenomenon of inverse relationship between age of bird and egg shell thickness.
Boulton et al. (1981) recorded a significant reduction in HI titers in layer breeders at
500ppb levels of AF.
Iqbal et al. (1983) fed the White Leghorn layers up to 5.00ppm dietary AF for three
periods each consisting of 28 days. They reported that feeding 1.00ppm level of AF resulted in
a significant reduction in hen day egg production and 2.00ppm level onwards feed efficiency
was adversely affected. Congested and haemorrhagic livers, enlarged spleens, and immature
ova with congestion were commonly seen. However, none of the levels affected feed
consumption, body weight, egg weight, shell percentage, Haugh unit scores and serum protein
levels. According to Dalvi and McGowan (1984), chronic AF toxicity in birds was
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characterized by drop in egg production. Washburn et al. (1985) reported that dietary AF at
5.00ppm fed for three weeks had no detrimental effect on shell strength but egg weight was
significantly reduced.
Johri and Sadagopan (1989) reported a significant reduction in hen day egg production
of laying quails when fed with 0.50 or 0.75ppm AF. Johri et al. (1990) studied the effect of low
levels of dietary AF (0.00-0.75ppm) in Japanese quail fed toxic diet for 100 d and reported that
egg production, protein utilization and body weight were adversely affected by 0.50 and
0.75ppm, whereas feed consumption and hatchability of fertile eggs were adversely affected by
0.30ppm. At 0.75ppm level, fertility of eggs and serum total protein decreased and serum
glutamic pyruvic transaminase (ALT) increased.
Aflatoxin when added at 0 and 10ppm, with tryptophan to a layer ration, showed
significant reduction in egg production percentage (Rogers et al., 1991). Rao and Joshi (1993)
included 1.25, 2.50, 5 and 10ppm AFB1 in layer rations for four weeks and found decreased
egg production in birds receiving 5 and 10ppm of AFB1.
Fernandez et al. (1994) reported a significant reduction in egg production and oral
lesions in layer chicken treated with 120ppb onwards for varying periods.
Azzam and Gabal, (1998) reported a significant reduction in egg production of
commercial layers fed with high levels of aflatoxin for six weeks.
Kubena et al. (1999) studied the effect of diets containing 50 or 100mg/kg
moniliformin fed to White Leghorn laying hens for 420 d and observed that egg production
was reduced by approximately 50 per cent by the end of the second 28-d laying period. Egg
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weights were reduced by the 100mg/kg toxin. The hens in toxin-treated group also had
significantly lower body weight than the other treatments. Mortality was minimal except in
hens fed with 100mg toxin/kg diet.
Mukhopadhy et al., (2000) have also reported a significant reduction in egg production
in commercial layers exposed to 500ppb aflatoxin given for 90 days.
Ginzberg et al. (2000) reported that only the yolk color in the group fed on 5 per cent of
Spirulina algae was 2.4 times darker compared to the control laying hens.
Nimruz (2002) found that yolk color index of layers was significantly improved by the
addition of Spirulina in feed. He concluded that Zeaxanthin content in the yolk tended to
increase significantly with the dosage of Spirulina.
Kim et al. (2003) found reduction in serum calcium, phosphorous and ALT and
increase in GGT levels in laying hens by dietary levels of 500ppb of AF given from week 67 in
laying hens.
Chowdhury and Smith (2004) reported decrese in feed efficiency when layers fed
Fusarium mycotoxins contaminated diets compared with control groups.
Ogido et al. (2004) reported an increase in feed consumption and decrease in egg
weight in Japanese quails fed with combination of 50ppb of AFB1 and 10ppm of fumonisin B1
for 140 d.
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Verma et al. (2004) reported decrease in hen day egg production, egg weight, feed
consumption, shape index, albumen index and Haugh unit due to feeding 1ppm of AFB1 for 42
d to White Leghorn hens aged 42 weeks.
Svetlana Grigorova (2005) reported that when adding 2 per cent and 10 per cent of dry
biomass from fresh water algae of Chlorella genus in the combined forages for laying hens, the
yolk pigmentation became significantly more intensive by 2.5 units by the Roche’s scale.
Ninety-six laying hens fed with 2.50ppm of AFB1 for four weeks by Zaghini et al.
(2005) showed decrease in egg weight, egg shell weight and increased protein percentage in
albumen. They reported that AF influenced color parameters, which was attributed to
interference of AFB1 with lipid metabolism and pigmentary substances deposition in yolk.
Further, no AFB1 or AFM1 residues were found in eggs of the experimental groups.
Pandey and Chauhan (2007) reported that feeding of AFB1 at the dose rate of 2.50,
3.13, 3.91mg/kg to the White Leghorn layers from first week to 40 weeks of age did not affect
the body weight but resulted in decreased feed consumption, reduction in both egg production
and egg weight at 3.91mg/kg level and caused 11-47 per cent dose-dependent mortality. They
also reported that feeding AFB1 at the dose rate of 2.50, 3.19 and 3.91mg/kg to the White
Leghorn layers resulted in paleness of breast muscles, discolored livers, enlarged and pale
kidney. Enlarged hearts and lungs were noticed at 3.13 and 3.19mg/kg levels. However, there
were no changes in the intestine and spleen at all levels, but the Bursa of Fabricius was
oedematous and enlarged at 3.91mg/kg level. Lymphoid depletion and lymphocytolysis and
reticuloendothelial cell hyperplasia in the spleen were also observed in all the toxin fed groups.
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Denli et al. (2008) reported a reduced daily feed consumption, egg mass, and serum
triglyceride concentrations, while increase in the relative liver weight, the serum activity of
alkaline phosphatase, and the serum concentration of uric acid in twenty-eight Hisex Brown
laying hens of 47 weeks of age fed with ochratoxin A for 3 weeks when compared those fed
with the control diet.
Thapa, 2008 reported a significant reduction in egg production of layers fed with
varying levels of aflatoxin for three periods.
2.5.2. Aflatoxicosis in breeders
When AF (20.00ppm) was incorporated into feed of mature broiler breeder males for
four weeks, no alteration in spermatozoa counts, semen volume, or semen DNA, RNA or
protein content was recorded (Briggs et al., 1974).
Howarth and Wyatt (1976) fed broiler breeder hens 5 and 10ppm of AF in their diet for
four weeks and reported no reduction in fertility, whereas hatchability of fertile eggs declined
significantly from 95.00 per cent in the control to 68.90 and 48.50 per cent, respectively in 5
and 10ppm AF fed groups. Egg production decreased significantly during weeks three and four
after initiation of toxin feeding in hens fed with 10 and 5ppm AF, respectively. They also
observed enlarged fatty and friable liver and enlarged spleens by feeding AF at the dose levels
of 0.00, 5.00 and 10.00ppm. Further, they did not observe any latent effect of AF or its
metabolites on the performance of the surviving chicks hatched from broiler breeder hens, fed
with 0.00, 5.00 and 100µg/kg of AF for four weeks.
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Sharlin et al. (1981) reported decreased semen volume and testes weight and disruption
of the germinal epithelium in mature White Leghorn males fed with 20.00ppm AF for five
weeks. They also noticed decrease in feed intake and body weight. However, there was no
effect on per cent fertile eggs or per cent hatchability of fertile eggs from hens artificially
inseminated with spermatozoa from the treated males.
When laying hens and mature cocks were fed diets containing 8.10ppm AFB1 or
1.60ppm AFG1 for three weeks, egg production ceased. Histological examination of the ovaries
showed follicular atresia. On the contrary, no testicular lesions were seen in the males (Hafez
et al., 1982). Histological evidence of adverse effects of AF on the germinal epithelium of the
testes was reported in immature chickens dosed with 200µg of AF/day/chick for 35 days
(Mohiuddin, 1982).
Jayakumar et al. (1988) fed AFB1 at rate of 25µg/duck, daily for three months and
noticed reduced fertility and hatchability. Khan et al. (1989) injected 26.00, 81.00 and 216.00
ng/egg of AFB1 and reported that lethal dose was 216.00 ng/egg and it caused mortality of
chick embryo by the fourth day of incubation.
Tiwari et al. (1989) compared the hatchability of chicks hatched from AF containing
eggs and concluded that it was low in comparison to chicks hatched from AF free eggs.
Further, they studied the post-hatch performance of chicks hatched from AF containing eggs
and observed lower weight gains and impaired defense system in chicks fed on normal diet.
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In a study by Abdelhamid and Dorra (1990) where the maternal diet contained
100.00ppb of AF, citrinin or patulin for six weeks, the chicks had significantly higher weight
than the control.
Rao (1990) observed a drastic deterioration in semen quality of breeder cocks fed with
1.000ppm AF. The traits affected were semen volume, semen concentration, motility and
abnormalities.
Stephen et al. (1991) reported a significant drop in egg production in layer chicken fed
with 5.00 and 10.00ppm AF for three weeks.
Nelson-oritiz and Qureshi (1992) assessed single dose exposure of six day-old embryos
to 0.100, 0.500 and 1.00µl of AFB1 and concluded that rate of mortality of the embryos was
dose related. Chick embryos, administered different levels of AF or ochratoxin on the chorio-
allontoic membrane showed decreased weight and length. Further, abnormalities like everted
viscera, exposed brain, crossed beak, underdeveloped eyes and head and twisted limbs were
observed.
Bergsjo et al. (1993) reported chick developmental anomalies when laying hens were
fed diets containing 4.90mg of DON/kg of feed for 10 weeks.
Diaz and Sugahara (1995) reported that birds fed AF at 0.66 or 3.00µg/kg diet did not
show any adverse effect on chick performance.
Muthiah (1996) conducted an experiment to study the effect of graded dietary levels of
AFB1 (0.00, 0.50, 1.00 and 1.50ppm) on the reproductive performance of layer breeders. He
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reported that the sperm motility and concentration were not affected while the percentage of
sperm abnormality increased when AFB1 was included in the diet of breeder cocks. The feed
consumption was significantly decreased and egg production declined in proportion to the level
of AFB1 incorporation in the diets. There was no effect on fertility but hatchability was
affected. The chicks hatched from breeder hens, received graded levels of AF in their diets did
not show any effects on body weight, weight gain, mortality and feed consumption during the
0-8 weeks post-hatch performance period.
Cotter and Weinner (1997) reported lowered hatchability in broiler breeder hens fed
with four levels viz., 0.00, 308.00, 610.00 and 1834.00ppb of AF.
Brake et al. (1999) conducted an experiment by feeding diets with different levels of
diacetoxyscirpenol (DAS) (ranging from 0.00 to 20.00mg/kg) to broiler breeders between 67 to
69 wk of age. They observed no effect on egg production, when DAS was fed upto the level of
5.00ppm. Furher, theyhave demonstrated that feeding diets contaminated with 10.00 and
20.00mg of DON per kg of feed decreased the fertility in broiler breeder males, though there
was no difference in the volume of semen produced.
Brake et al. (2000) reported that there were dose-related decreases in body weight and
feed consumption indicating feed refusal, as well as dose-related increases in the extent of
mouth lesions of broiler breeders fed with 0.00, 5.00, 10.00, or 20.00mg DAS/kg diet from 24
to 25 wk of age.
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Stanley et al. (2004) reported that feeding AF at the rate of 3mg/kg to 35 week’s old
Cobb broiler breeder hens for three weeks significantly reduced serum total protein, albumin,
calcium and phosphorus levels.
Sypecka et al. (2004), reported that only trace amounts of Fusarium mycotoxins are
transferred into the eggs of laying hens, which are unlikely to be of significance with respect to
embryonic mortality.
Yegani et al. (2006) reported no effect in feed consumption, body weight, and egg
production. However, increase in early embryonic mortality (1to7d) in eggs from birds fed
contaminated grains with deoxynivalenol (12.60mg/kg of feed) was observed in broiler breeder
hens. They also reported that the ratio of chick weight to egg weight was not affected. Weight
gains of chicks fed a standard broiler starter diet at 7, 14, and 21 d of age were also not
significantly affected by previous dietary treatments for the dam. Feeding of contaminated
diets did not affect semen volume, sperm concentration, viability, and motility. There was no
effect of diet on the relative weights of liver, spleen, kidney and testes.
2.5.2.1. Aflatoxin residue in eggs
Although the concentration of mycotoxins and their metabolites are generally much
lower in eggs than in animal feeds and are not likely to cause acute intoxications in humans.
However, the residues of carcinogenic mycotoxins such as AFB1 and M1, (AFM1 is a polar
metabolite of AFB1) and ochratoxin A, when present in animal products are a threat to human
health and must be monitored. The limit for AFB1 in complete feeds is 0.02mg/kg.
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Trucsksess et al. (1983) were able to detect AFB1 and M1 residues in eggs of hens fed
contaminated feed. After 7 days of withdrawal only trace amounts remained in eggs.
According to Wolzak et al. (1985) clearance of AF occurs faster from the albumen than from
the yolk.
Aflatoxin and some of their metabolites can be carried over from feed to eggs in ration
ranging from 5,000:1 to 66,200:1 and even to 125,000:1, whereas in other trials no measurable
residual AFB1 or its metabolites were found in eggs (Oliveira et al., 2002).
Zaghini et al. (2005) reported that no traces of AFB1 or AFM1 residues were found in
eggs of layer hens supplemented with diet containing 2.50ppm AFB1.
In a recent study, Salwa and Anwer (2009) reported no traces of AF in the eggs of
layers fed with 25.00, 50.00 and 100ppb of AF in their diet for 60 days.
2.5.3. Aflatoxicosis in broilers
2.5.3.1. Effect on growth, feed consumption, mortality and organ weights
The growth retardation due to aflatoxicosis is well documented. The main causes for
the growth depression are reduced feed intake, altered protein metabolism, altered enzymatic
activity, decreased nutrient utilization and absorption. The effect of feeding various levels of
AF on growth, feed consumption, mortality and organ weights in broilers reviewed from
literature is summarized in Tables 2.2, 2.3, 2.4 and 2.5, respectively.
2.5.3.2. Feed conversion ratio
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Giambrone et al. (1985) reported that chicks fed a diet containing 1.10ppm AFB1 from
naturally contaminated moldy wheat had a poorer feed conversion than those of control.
Interestingly, no effects on feed conversion were noted when pure AFB1 was administered at
0.50 and 1.00ppm levels to 2 and 5 week old broiler chicken.
Feed efficiency (0-6weeks) in broilers fed with 200 and 400ppb AF was significantly
lower when compared to control (Swamy and Devegowda, 1998). Poorer feed efficiency (0-
5weeks) was observed in broilers when fed diets containing 0.30ppm AFB1 (Raju and
Devegowda, 2000). Arvind et al. (2003) observed 2.30 per cent reduction in feed efficiency on
feeding naturally contaminated diet containing 168ppb of AF to broilers. Verma et al. (2004)
also reported reduction in feed efficiency due to aflatoxicosis in broilers.
Miazzo et al. (2005) reported a significant poorer feed gain ratio of broilers fed
2.50ppm of AF and Gowda et al. (2008) reported no significant effect on broilers fed with
1.00ppm of AF.
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Table 2.2 : Effect of feeding various levels of aflatoxin on growth in broilers
Author and year Species and duration Level (ppm) Remarks
Huff et al. (1983) Male broilers (0-3 wks) 2.50 and 5.00 Significant reduction in body
weight
Phillips et al. (1988)
Broilers (0-3 wks) 7.50 Significant reduction in body
weight after 2nd week
Kubena et al. (1990)
Broilers (0-4 wks) 3.50 Significant reduction in body
weight by 8 days
Giroir et al. (1991) Broilers (0-3 wks) 1.50 Significant reduction in body
weight
Devegowda et al. (1994)
Broilers (0-6 wks) 0.25 Depressed growth in a dose
dependent manner
Swamy and Devegowda (1998)
Broilers (0-6 wks) 0.10 Significant growth depression
Santurio et al. (1999)
Broilers (4 days)
0, 0.10, 0.20 and 0.40
Growth depression from 0.2 ppm onwards
Raju and Devegowda (2000)
Broilers (0-5 wks) 0.30 Reduced body weight
Arvind et al. (2003)
Broilers (0-5 wks) 0.16 Depressed growth
Girish and Devegowda (2004)
Broilers (0-5 wks) 2.00 Depressed growth
Miazzo et al. (2005)
Broilers (0-5wks) 0.50 Significantly diminished body
weight
Manafi, (2006) Broilers (0-5wks) 0.50 Significant reduction in body
weight
Gowda et al. (2008)
Broilers (0-3wks) 1.00 Reduced body weight
significantly
39
Table 2.3 : Effect of feeding various levels of aflatoxin on feed consumption in broilers
Author and year Species and duration Level (ppm) Remarks
Dalvi and McGowan (1984)
Broilers (0-8 wks)
0, 2.50, 5.00 and 10.00
Dose dependent reduction in feed consumption
Kubena et al. (1990)
Broilers (0-4 wks) 3.50 Reduced feed intake
Giroir et al. (1991) Male broilers (0-3 wks) 2.50 Significant decrease in feed
consumption Raju and
Devegowda (2000) Broilers
(0-5 wks) 0.30 Reduced feed consumption
Arvind et al. (2003)
Broilers (0-5 wks) 0.16 Decreased cumulative feed
intake
Verma et al. (2004) Broilers (0-7wks)
0.50,1.00 and 2.00
Significant decrease in feed consumption
Miazzo et al. (2005)
Broilers (0-5wks) 0.50 Significantly decreased feed
consumption Gowda et al.
(2008) Broilers (0-3wks) 1.00 Significant decrease in feed
consumption
Table 2.4 : Effect of feeding various levels of aflatoxin on mortality in broilers
Author and year Species and duration Level (ppm) Mortality
Asuzu and Shetty (1986)
Broilers (0-2 wks) 2.65 More than 25% of the total
birds died
Kubena et al. (1990) Broilers (0-21days) 3.50 10%
Rajendra (1993) Broilers (0-5 wks) 0.50 and 1.00 7 % and 19 %, respectively
Swamy and Devegowda (1998)
Broilers (0-6 wks) 0.40 5%
Raju and Devegowda (2000)
Broilers (0-5 wks) 0.30 3.33 %
Bhaskar et al. (2003) Broilers (0-6 wks) 0.20 23.33%
Girish and Devegowda, (2004)
Broilers (0-5 wks) 2.00 5%
Manafi, (2006) Broilers (0-5wks) 0.50 16.67%
40
Table 2.5 : Effect of feeding various levels of aflatoxin on organ weight in broilers
Author and year Duration of exposure Level (ppm) Remarks
Phillips et al. (1988)
Broilers (0-3 wks) 7.50 Friable, enlarged and pale livers
Kubena et al. (1990)
Broilers (0-4 wks) 3.50 Increase in relative weights of
all internal organs
Espada et al. (1992) Chickens (0-4 wks) 0.50
Pale yellow livers, edema of gall bladder, multi focal areas of
congestion in kidneys
Sundaresan and Mani (1996)
Broilers (0-5 wks)
0.10, 0.20, 0.30, 0.40 and 0.50
Increase in liver weight with bursal regression
Swamy and Devegowda (1998)
Broilers (0-6 wks) 0.1, 0.2 and 0.4
Enlarged, friable, pale and congested livers. Increased relative weights of liver and gizzard in 0.4 ppm fed birds
Raju and Devegowda, (2000)
Broilers (0-5 wks) 0.30 AF significantly increased both
liver and kidney weights
Arvind et al. (2003) Broilers (0-5 wks) 0.16 Increased relative weight of
liver and gizzard
Girish and Devegowda (2004)
Broilers (0-5wks) 2.00 Increase in weights of liver,
kidney, gizzard and spleen
Gowda et al. (2008) Broilers (0-3 wks) 1.00 Increased relative weight of
liver and gizzard
2.5.3.3. Gross lesions
Ducklings and chicks showed inappetence, poor growth rate and mortality at the age of
two weeks on feeding with AF contaminated groundnut meal. Necroscopy examination
revealed liver enlargement, pale buff coloration with superficial hyperplastic nodules,
occasionally pin head size foci and petechial haemorrhages (Asplin and Carnaghan, 1961).
Archibald et al. (1962) reported paleness and enlargement of liver and kidneys on feeding
different levels of AF ranging from 0.20 to 0.50ppm in broilers.
41
Tung and Hamilton (1973) reported significantly enlarged liver and kidney in broilers
fed with graded levels of aflatoxins ranging from 0.625 to 10.00µg/gm of feed. Huff et al.
(1988) reported similar effect by feeding 2.50 and 5.00ppm of AF in broilers. Phillips et al.
(1988) reported that the liver from chicks fed with diets containing 7.50mg/kg of AFB1 were
friable and pale in appearance. Similar findings were reported by Rajendra (1993) and
Subramanya (1993) by feeding AF at 0.50, 1.00ppm and 0.25 and 0.50ppm to broilers. Miazzo
et al. (2005) reported that liver of birds fed diets containing 2.50ppm of AFB1 were enlarged,
yellowish, friable, and had rounded borders.
Gowda et al. (2008) reported that lipid peroxide level was increased in liver
homogenate of chicks fed with 1.00ppm of AFB1.
2.5.3.4. Plasma proteins
Aflatoxicosis adversely affects the concentration of serum constituents, total serum
protein, globulin, albumin, cholesterol and uric acid. Among them, total serum protein and
albumen are the ones which are most affected. The reduction in total serum protein is due to
the impairment of amino acid transportation at mRNA transcription level and there by
inhibiting protein synthesis (Thaxton et al., 1974).
Aflatoxin B1 is metabolized in the liver which is having a high level of metabolizing
enzymes and induces damage to this organ even leading to hepatocarcinogenesis (Hsieh,
1985). Maurice et al. (1983) observed significant reduction in plasma proteins when broilers
were fed with 50 to 100ppb of AFB1 through oral route for a period of 0 to 3 weeks.
42
Mert et al. (1990) recorded decreased total protein concentration in serum by feeding
5.00µg of AFB1 daily for a period of two months. Decreased serum protein content by AF
feeding was also reported by Umesh et al. (1990) and Devegowda et al. (1994).
Feeding diets with AF to the broiler chickens resulted in significant increase in
creatinine levels (Abdelhamid et al., 1994).
Swamy and Devegowda, (1998) substantiated the depressing effects of AF on
cholesterol (12.5 to 57.15 per cent).
2.5.3.5. Serum enzyme activity
The increase in serum enzyme levels noted during aflatoxicosis can be interpreted as
the sequelae of hepatocyte degeneration or damage to the cell membrance and subsequent
leakage of enzyme into the circulation (Devegowda et al., 1994).
Altered activities of enzymes i.e. alanine amino transferase (ALT), aspartate amino
transferase (AST), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), and many
other enzymes have also been noticed during aflatoxicosis. Among these, GGT is the most
sensitive indicator of aflatoxicosis (Arshad et al., 1993). In several other studies, increased
levels of AST and ALT were observed (Mani et al., 2000).
Jindal et al. (1993) reported significantly higher levels of ALT and ALP activities in
broilers fed with 500ppb AF. However, Huff et al. (1988) could not find any alteration in
serum alkaline phosphatase activity in AF fed broilers.
43
Kumar et al. (1993) found an increase in serum alkaline phosphatase level when broiler
quail chicks were fed AF at the rate of 0.50, 1.00, 1.50 and 2.00ppm for 3 weeks.
Raju and Devegowda (2000) reported significant increase in serum levels of GGT upon
feeding of 300ppb AFB1 for 35 days in broilers. Nataraj et al. (2004) reported significant
decrease in serum total protein, albumin, phosphorus and calcium levels in broilers fed with
AF. Gowda et al. (2008) reported increased GGT levels during exposure of broilers to 1.00ppm
of AFB1 for 3 weeks.
2.5.3.6. Immune system and antibody response
The most important outcome of aflatoxicosis in poultry was impairment of immune
system resulting in high mortality. AF inhibits DNA and protein synthesis through impairment
of amino acid transport and m-RNA transcription, resulting in lowered level of antibody
production (Thaxton et al., 1974). Poor immune response to Newcastle disease vaccination
during aflatoxicosis was reported by Chenchev et al. (1978).
Though the exact mechanism is not known, it is believed that the impaired
immunogenesis was by suppressing the formation of nonspecific humoral substances related to
resistance and immunity, suppressing phagocytolysis by macrophages and causing thymic
aplasia (Pier and McLaughlin, 1985).
Broilers fed with diets containing as low as 250 and 500ppb AF have shown significant
reduction in HI titer values of Newcastle disease (ND) virus antibodies (Devegowda et al.,
1994). A significant reduction in the size of bursa was seen in broilers fed 500ppb AF
44
(Devegowda et al., 1994). However, Swamy and Devegowda, (1998) did not notice any
significant reduction in bursa and spleen weights by feeding AF up to 400ppb.
Decreased antibody titers against ND and Infectious Bursal disease (IBD) were
reported in broilers fed with AF (Umesh et al., 1990; Devegowda et al., 1994; Swamy and
Devegowda, 1998; Pasha et al., 2007).
In the majority of animal species, resistance to infection was reduced by simultaneous
exposure to AFB1. Effect of AFB1 was primarily on the cell-mediated immune functions.
However, T cell dependent humoral responses were also adversely affected (Swamy and
Devegowda, 1998).
Miazzo et al. (2005) reported a significant increase in spleen weight of broilers fed with
500ppb of AFB1 contaminated diet for 5 weeks. Girish et al. (2008) reported an increase in
weights of bursa and spleen of turkeys fed with Fusarium mycotoxin for 12 weeks.
2.6. Counteraction of aflatoxicosis
The infestation of agricultural products, intended for human and animal consumption
with toxigenic fungi that are capable of producing highly toxic metabolites has been a
worldwide problem. Increased efforts are being undertaken to develop cost effective and safe
procedures and products to effectively deal with the decontamination and remediation of
mycotoxin contamination in feedstuffs. The available approaches were reviewed by Trenholm
et al. (1996) and Devegowda et al. (2003).
The methods aimed at preventing or reducing the level of mycotoxin contamination
were classified as preventive or curative. The following approaches were recommended:
45
1. Prevention of the initial growth of moulds and subsequent production of mycotoxin.
2. Detection of mycotoxin in feed and selective removal of contaminated portions.
3. Inactivation or destruction of the toxin by physical, chemical and biological means.
4. Utilization of mycotoxin resistant genetic resources.
2.6.1. Physical methods
According to Park and Liang, (1993) Mycotoxins (AF, ochratoxin, T-2 toxin and
citrinin) are highly soluble in organic solvents. Their extraction from the feed stuffs using
several solvents or mixture of solvents has been proved to be highly effective.
Scott (1989) opined that thermal treatment appears to have little effect on the toxin
content as mycotoxins are heat resistant. Irradiation of feed stuffs may reduce the toxin content
considerably. Exposure of contaminated feed ingredients to sunlight may also prove to be
effective. These methods have little practical applicability.
The utilization of mycotoxin-binding adsorbents, which do not get absorbed from the
GIT and instead bind physically with mycotoxin, is the most applied physical method of
protecting animals against the harmful effects of mycotoxin contaminated feed and has gained
considerable attention in recent times. The efficiency of the adsorption depends on the
chemical structure of both the adsorbent and the mycotoxin. Before applying this technique for
routine use, it is essential to establish that the adsorbent does not remove essential nutrients
from the diet.
Clays are made of two or more mineral-oxide layers. These layers are stacked parallel
units of silica and alumina sheets. The silica form tetrahedral sheets and the alumina forms
46
octahedral sheets. Some of these clay particles have the ability to absorb moisture and will
expand while others do not. The difference is due to clay chemistry and the elements (cations)
that are components of the layers.
A brief explanation of most used adsorbents (hydrated sodium calcium
aluminosilicates, activated carbon, bentonite, clays and special polymers) is summarized in the
following headings:
2.6.1.1. Zeolites
Zeolites are crystalline aluminosilicate compounds that are classified according to
common features of the framework structures. Particularly, the zeolite structure known as type
A has a specific arrangement in which the unit cell contain 24 tetrahedra, 12 AlO4, and 12
SiO4, when fully hydrated (Sivapullaiah et al., 2000). Feeding spent canola oil bleaching clays
and cholestryamine was useful in alleviating the adverse effects of T-2 (Dvorak, 1989). An
improvement in body weight and reduction in the relative weight of liver was reported in
broilers exposed to 2.50ppm AF by dietary supplementation of one per cent synthetic zeolites
(Miazzo et al., 2000). An improvement of 29 to 41 per cent in body weight gain was reported
in broilers exposed to 3.50ppm AF by dietary supplementation of commercial zeolite (Duarte
and Smith, 2005).
2.6.1.2. Hydrated sodium calcium, aluminosilicate (HSCAS)
Hydrated sodium calcium aluminosilicate, a phyllosilicate derived from natural zeolite,
is perhaps the most extensively investigated sorbent. Phillips et al. (1988) showed that
hydrated sodium calcium aluminosilicates (HSCAS) have high affinity for AFB1 after
47
screening 38 different adsorbents that were representative of the major chemical class of
aluminas, silicas and aluminosilicates. Incorporation of hydrated sodium calcium
aluminosilicate (HSCAS) at 0.5 per cent level in diet had been reported to reduce the toxicity
of AF (Phillips et al., 1988; Kubena et al., 1990; Huff et al., 1992; Jindal et al., 1993).
However, HSCAS have little effect on the toxicity of ochratoxin and T-2 toxin in broilers
(Kubena et al., 1990; Huff et al., 1992; Raju and Devegowda, 2000; Girish and Devegowda,
2004; Duarte and Smith, 2005; Schollenberger et al., 2006).
2.6.1.3. Activated carbon
Activated carbon (AC), an insoluble powder formed by pyrolysis of different kinds of
organic materials, showed different adsorbing properties depending on its origin. Surface area
of activated carbons may vary from 500 to 2000 m2/g and up to 3500 m2/g for super active
carbons. AC is quite effective for adsorbing OA from aqueous solution, but had no beneficial
effect when tested in vivo (Rotter et al., 1989). In another experiment, Barmese et al. (1990)
observed an improvement in body weight of broilers on inclusion of 0.2 per cent activated
charcoal in the diet containing 0.50ppm of AF. Beneficial effects of AC have been shown in
rats intoxicated with T-2 toxin. The mechanism of this beneficial effect has been associated
with the ability of the AC to bind the mycotoxin, preventing it’s absorption and especially
enterohepatic recirculation (Ramos and Hernandez, 1997; Solfrizzo et al., 2001; Duarte and
Smith, 2005).
2.6.1.4. Bentonite
Bentonite, a clay mineral of the smectite group is formed of highly colloidal, plastic
clays, composed of mainly montmorillonite and is produced by in situ diversification of
48
volcanic ash. Bentonite may also contain feldspar, biotite, kaolinite, illite, cristobalite,
pyroxene, zircon, and crystalline quartz (Parker and McGraw-Hill, 1988). It has the unique
characteristic of swelling to several times its original volume when placed in water and of
forming thixotropic gels with water even when the amount of clay is relatively less.
Based on interchangeable action, composition, bentonites can be classified as calcium,
magnesium, and potassium or sodium bentonites. Na-bentonite has greater swelling property
and plasticity than Ca-Bentonite. While Ca-Bentonite is most common, Na-Bentonite is found
in few localities (Hanchar et al., 2004).
2.6.1.4.1. Physical and chemical properties
Bentonite feels greasy and soap-like to touch (Bates and Jackson, 1987). Freshly
exposed bentonite is white to pale green or blue and on exposure darkens in time to yellow,
red, or brown (Parker and McGraw-Hill, 1988).
Interstitial water held in the clay mineral lattice is an additional major factor controlling
the plastic, bonding, compaction, suspension and other properties of montmorillonite-group
clay minerals. Within each crystal, the water layer appears to be an integral number of
molecules in thickness. Physical characteristics of bentonite are affected by whether the
montmorillonite compose has water layers of uniform thickness or whether it is a mixture of
hydrates with water layers of more than one. Loss of absorbed water from between the silicate
sheets takes place at relatively low temperatures (100–200°C). Loss of structural water (i.e. the
hydroxyls) begins at 450–500°C and is complete at 600–750°C.
49
Further heating to 800–900°C disintegrates the crystal lattice and produces a variety of
phases, such as mullite, cristobalite, and cordierite, depending on initial composition and
structure. The ability of montmorillonite to rapidly take up water and expand is lost after
heating to a critical temperature, which ranges from 105 to 390°C, depending on the
composition of the exchangeable cations. The ability to take up water affects the utilization and
commercial value of bentonite (Parker and McGraw-Hill, 1988).
Montmorillonite clay minerals occur as minute particles, which, under electron
microscopy, appear as aggregates of irregular or hexagonal flakes or, less commonly, of thin
laths (Grim, 1968). Chemical properties of bentonite are detailed here under:
Parameters
Moisture (%) ( loss on drying at 110 °C for 3 hours) 10-12
pH (at 25 °C) (5 % suspension) 3-4
Average Particle size (microns) 5-10
90% Passing (size in microns) 40
BET surface area 340-360
For a clay particle to adsorb or bind to an organic molecule such as a feed mycotoxin
there must be opposite electrical charges that attract. Clays with a high cation exchange
capacity (CEC) have a large number of negative charges on their surfaces. Those clays with
intermediate or low CEC have mixed positive and negative charges.
Clay particles may be electrically neutral with an equal number of positive and negative
charges. Since AFB1 has been shown to bind with aluminosilicates or bentonites that contain a
large number of negative charges (high CEC), the AF molecule must contain positive charges
or be able to absorb a positive charge. Several of these clay products do not bind to mycotoxins
50
other than AF, which may be due to the polarity of the electrical charges on the clay particles
to the location of those electrical charges or the sequence of the locations on the clay surface.
For secure binding to occur, it may take multiple electrical sites to hold the mycotoxin
molecule even when the correct electrical charges are present. The surface shape of the clay
particles, pore size and acidity (pH) may effect the binding as well. More chemically reactive
organic molecules (free-radicals) would be expected to form more stable bonds than lower
reactive molecules. The clays have several different types of reactions which can cause binding
of minerals or organic molecules to the clay particles. Protonation is one type, where the
organic molecule accepts a proton to become basic. As water content decreases, proton
donating abilities of the clay surface increases. One of the major companies in this field has
tested many clay products and found wide differences in their ability to bind specific
mycotoxins. The only data of importance to the user of these types of products is the animal
test data on a specific product for its ability to bind a specific mycotoxin.
2.6.1.4.2. Surface chemistry
Chemical properties of the surfaces of silicates and, in particular, clay minerals are
strongly dependent on their mineral structure. The basic unit of montmorillonite crystals is an
extended layer composed of an octahedral alumina sheet (O) between two tetrahedral silica
sheets (T), forming a TOT unit (Bailey et al., 1998). The stacks of TOT layers produce the
montmorillonite crystals. Isomorphic substitutions in the octahedral sheet (few tetrahedral
substitutions are observed in montmorillonite) create an excess of negative structural charge
that is delocalized in the lattice. The external and interlayer surface area represents
approximately 95 per cent of the total surface area of montmorillonite. On the other hand, the
periodic structure of the montmorillonite crystals is interrupted at the edges, where the broken
51
bonds compensate their charge by the specific adsorption of protons and water molecules
(Schindler and Stumm, 1987; Stumm and Wollast, 1990; Stumm, 1997). This interruption of
the periodic structure confers to the edge surface an amphoteric character i.e. a pH-dependent
surface charge and the capacity to react specifically with cations, anions, and molecules
(organic and inorganic) forming chemical bonds. The chemical composition of bentonite is as
follows:
Per cent on dry basis
SiO2 71.64
Al2O3 10.22
Fe2O3 3.88
TiO2 0.72
CaO 1.47
MgO 1.48
P2O5 0.01
Na2O5 0.38
K2O 0.049
SO3 0.97
Loss on ignition at 1000°C for 1 hour (Moisture free sample) 9.04 (Source: Manafi, 2006)
2.6.1.4.3. Uses
Bentonite has many applications with wide range of industrial and other activities.
Major uses of bentonite include serving as an ingredient for ceramics; waterproofing and
sealing in civil engineering projects, as a carrier in pesticides and fertilizers, and as a binding
52
agent in animal feeds (Patterson and Murray, 1983; Hosterman and Patterson, 1992; Hanchar et
al., 2004).
Swanson and Corley (1989) reported that bentonite improved growth rate and feed
efficiency in broilers. Further, they demonstrated improved energy and protein inhibition and
prolonged food passage time in the presence of bentonite.
Clay materials have the capability to bind molecules of certain sizes and configurations
and have been used effectively to decrease effects of AF contaminated grains in poultry. It is
postulated that bentonite formed a complex with the toxin thus preventing the absorption of AF
across the intestinal epithelium.
Unsworth et al. (1989) reported improvement in growth rate of broilers when sodium
bentonite, a natural clay was included in AF contaminated diet at 0.5 and 0.1 per cent.
However, the ability of bentonite to bind to mycotoxins depends on pH, molecular
arrangements and its geographic region of origin (Vieira, 2003).
Bentonite when included at 10 per cent was the most effective treatment for feed
refusal and growth depression of rats caused by 3.00ppm of T-2 toxin for 2 weeks (Smith and
Ross, 1991). Improvement in body weight at 7 and 14 days and increase in feed consumption
in broilers fed T-2 toxin with one and 2 per cent of sodium bentonite has been reported by
Hagler et al. (1992).
Santurio et al. (1999) evaluated the protective effects of bentonite in the prevention of
aflatoxicosis and concluded that sodium bentonite partially neutralized the effect of AF in
broiler chicks when included at 5.00g/kg in the diet. In a similar study, Rosa et al. (2001)
53
reported moderate protective effect of 0.3 per cent bentonite against the development of
aflatoxicosis in broilers.
Eralsan et al. (2005) reported a moderate increase in the albumin: globulin ratio of
broilers by addition of 0.3 per cent hydrated sodium bentonite in AF mixed feed of broilers.
They also reported that histopathological finding in liver sections of broilers fed AF plus
hydrated sodium bentonite indicated no protective effect of this adsorbent.
Due to their montomorillonite content, bentonites swell and form thixotropic gells as
result of their ion exchange capabilities and are widely used as mycotoxin sequestering agent
(Duarte and Smith, 2005). Eralsan et al. (2006) reported the effectiveness of sodium bentonite
in relieving the damages due to the presence of aflatoxin (1.00ppm) in 45 day old broiler
chicken.
2.6.1.5. Other adsorbent compounds
Divinylbenzene-styrene polymers (anion-exchange resins) exhibited beneficial effects
when added to the diet of T-2 intoxicated rats, minimizing the reduction in feed consumption
and the growth-depressing effect caused by T-2 toxin. The addition of divinylbenzene-styrene
to diets of rats supplemented with zearalenone resulted in a major decrease in urinary excretion
of conjugated zearalenone and its metabolites (Ramos et al., 1996). Polyvinylpyrrolidone (0.2
per cent) added to the diets of pigs contaminated with deoxynivalenol did not appear to
alleviate the toxic effect of this toxin when fed to barrows and gilts over a period of 5 weeks
(Ramos et al., 1996).
54
2.6.2. Herbal methods
2.6.2.1. Antioxidant agents
Application of some herbal extracts of plant origin like turmeric (Curcuma longa),
garlic (Allium sativum) and asafetida (Ferula asafetida) have shown to counteract aflatoxicosis
in animals and poultry through their antioxidant activity.
According to Surai (2001) and Weiss (2002) the antioxidant system in the body mainly
involves reducing agents (tocopherol, ascorbic acid, glutathione, carotenoids), peroxidases
(glutathione peroxidase, catalase), enzymes (peptidases, proteases, vitamin A) and superoxide
dismutase (SOD). The most common functional chemical groups which have radical
scavenging properties are hydroxyl (phenolics), sulfhydryl (cysteine, glutathione), and amino
groups (uric acid, spermine). Antioxidative phenolics include tocopherol, catechins,
ubiquinone and synthetic compounds (Butylated hydroxyanisole, Butylated Hyrdoxytoluene).
The antioxidant activity of phenolics is influenced by alkyl and hydroxyl groups which
can enhance their reactivity to neutralize lipid radicals. In processed food and/or feed stuff, the
antioxidant property is influenced by heat stability and pH variation. All antioxidants are not
suitable for addition to diets due to stability concerns, solubility, and interaction with other
feed components (Shahidi and Wanasundara, 1992).
There is sufficient evidence to suggest that antioxidants ameliorate oxidative stress
during mycotoxicosis by reducing the level of free radicals. Several natural (vitamins,
55
provitamins, carotenoids, polyphenols, micronutrients) and synthetic compounds seem to be
chemo protective against common mycotoxins. However, most data available on the above
aspect are from in vitro studies. Hence, it is warranted for more in vivo research with emphasis
on the level of supplementation vis–a–vis protection as higher levels of some antioxidants
could well be toxic to cellular systems (Atroshi et al., 1999; Renzulli et al., 2004). Plant
components such as coumarins, flavonoids, galangin and curcuminoids have been shown to
inhibit the metabolic biotransformation of aflatoxins to their hepatotoxic/carcinogenic epoxide
derivatives (Lee et al., 2001). The decline in antioxidant enzyme activity in oxidative stress is
due to reduction in protein biosynthesis and inhibition of RNA synthesis, and DNA dependent
RNA polymerase activity (Gowda and Ledoux, 2008).
2.6.2.2. Vitamin and related substances
Vitamin E occurs in 8 different forms, which vary greatly in their degree of biological
activity. Ascorbic acid, α–carotene, selenium, uric acid and vitamin E exhibited
anticarcinogenic effects in liver of rats administered with AFB1 (Nyandieka et al., 1990).
Supplementation of vitamin A, C and E in excess of 25 per cent of their requirement in the
diets reduced the negative effects of AFB1 in turkeys. Coelho (1996) reported that α-carotene
acts as an efficient scavenger of singlet oxygen (1O2), can neutralize peroxy radicals with
greater efficiency than of ά-tocopherol and vitamin A also has similar antioxidant properties.
According to Hoehler and Marquardt (1996) supplementation of vitamin E partially
ameliorated the prooxidant effects of ochratoxin A and T-2 toxin but not the vitamin C. There
are reports of regeneration of ά-tocopherol on reaction with other reducing agents like
glutathione and urate (Kagan and Tyurina, 1998) and ascorbate (May et al., 1998). Chow
(2001) stated that α-tocopherol is the most biologically active form, with quickly scavenges
56
peroxy radicals by forming a stable tocopheroxy radical and acts as a biological modifier.
Vitamin E pretreatment significantly lowered AF induced lipid peroxidation in testes of mice
(Verma and Nair, 2004). Among the vitamins, ascorbate (vitamin C) is most important because
of it’s ability to scavenge superoxide, hydrogen peroxide, hydrogen radical, hypochlorous acid,
and singlet oxygen (Chow, 2001). Riboflavin also has a protective action against AFB1 induced
DNA damage in rats. Glutathione, a cellular antioxidant plays an important role in free radical
scavenging and detoxification of electrophilic intermediates capable of lipid peroxidation
(Gowda and Ledoux, 2008).
2.6.2.3. Herbal compounds
Several herbal products contain antioxidant substances capable of scavenging free
radicals and enhancing antioxidant enzymes. Nyandieka et al. (1990) reported that use of
ethanolic extract of Cassia senna, (herb) as laxative inhibited the mutagenic effects of AFB1.
Feeding of the extract of Azadirachta indica prevented metabolic activation of AFB1 to its
epoxide derivative. Hepatic antioxidant status of rats was enhanced by feeding of a phenolic-
lignin enriched extract of the fruit Schisandra chinensis and provided hepato protection against
AFB1.
Oxidative changes (increased peroxides, reduced antioxidant enzyme activity) in liver
and kidney due to AFB1 were reversed in rats by feeding a root/rhyzome extract of Picrorhiza
kurroa (Picroliv) and a seed extract of Silybum marianum (Silymarin) (Weiss, 2002).
Similarly, Rosamarinic acid, a phenolic component of Boragnaceae species of plants (sage,
basil, and mint) reduced free radical oxygen formation, and inhibited protein/DNA synthesis as
well as apoptosis of human hepatoma cells caused by AFB1 and ochratoxin A.
57
According to Gowda and Ledoux (2008), ellagic acid, a phenolic compound of
strawberries and grapes showed anticarcinogenic activity and inhibited AFB1 mutagenicity. S-
methyl methane thiosulfonate present in cabbage and onion suppressed chromosomal
aberrations due to AFB1 in rat bone marrow cells. Diterpenes, cafestol and kahweol present in
green and roasted coffee beans prevented the covalent binding of AFB1 to DNA by modulation
of the carcinogenesis enzyme system. Further, they have listed antioxidants present in various
plants as detailed in Table 2.6.
Iqbal et al. (1983) observed chemoprotective effect of piperine (1-piperoyl piperidine),
an alkaloid of pepper against AF by inhibiting cytochrome P 450 bioactivation of AFB1. The
protective effect of chlorophylline (a derivative of the green pigment chlorophyll) against
AFB1 was also observed. The toxic effects of AF in chicken was reversed by the administration
of an alcoholic extract of African nut meg. The carbonyl functional groups of the curcuminoids
are thought to be responsible for their antimutagenic and anticarcinogenic action. Further,
strong inhibitory effect of curcumin on superoxide anion generation was noticed.
Table 2.6 : List of antioxidants present in various plants
Plant Antioxidant
Grape skin (Vitis vinifera) Phenols/anthocyanins
Sweet clover (Melilotus officinails) Coumarin
Green tea (Camellia sinensis) Catechins
Sorghum (Sorghum biocolor) Phenols
Oregano (Oreganum vulgare) Quercetin, galangin, carvacrol, thymol, rosamarinic acid
Coriander (Coriandrum sativum) Linalool
Cumin (Cumin cynicum) Cumin aldehyde
Fennel (Feoniculum vulrare) 3-caffeoylquinic acid, rosamarinic acid,
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quercetin-3-O-galactoside
Ginger (Zingiber officinale) Gineol, diarylheptanoids
Nutmeg (Myristica fragrans) Terpene hydrocarbons (limonene, myrcene, linalool, gerniol, terpineol, safrole)
Black pepper (Piper nigrum) Piperine
Chilli peppers (Capicum annuum) Capsaicin, flavonoids
Cinnamon (Cinnamonum verum) Cinnamic acid aldehyde
Clove (Syzgium aromaticum) Eugenol, eugenyl acetate
Pomegranate peel (Punica Granatum pericarpium)
Polyphenolics
Yucca bark (Yucca schidigera) Resveratrol
Turmeric rhyzome (Curcuma longa) Curcuminoid pigments Source: Gowda and Ledoux (2008)
Certain hormonal substances like melatonin also have strong antioxidant action through
free radical scavenging and protection of nuclear DNA and membrane lipids from oxidative
damage. Melatonin reduced the production of peroxide in rats fed with diets containing AFB1
and ochratoxin A. The oils of black cumin (Nigella sativa) and clove (Syzygium aromaticum)
prevented the toxic effects of AFB1 in rats (Gowda and Ledoux, 2008).
The compounds imparting the yellow color to turmeric powder are curcumin (1, 7, bis-
4-hydroxy-3-methoxyfenil-1, hepatadiene-3, 5-dione) and the curcuminoids (Demethoxy
curcumin and bis–demethoxy curcumin). Supplementation of turmeric (curcumin) to AF
intoxicated ducklings was effective in reducing the liver damage through reduction in AFB1-
DNA adducts formation, and modulation of cytochrome P 450 activity. Addition of turmeric
powder (0.5%) containing 1.4 per cent of total curcuminoids to an AFB1 contaminated chick
diet increased the activity of SOD and reduced the peroxide leveling homogenates of broiler
chicks (Gowda et al., 2008).
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2.6.2.4. Spirulina Platensis
It has been reported that some species of algae are also effective against aflatoxicosis.
Spirulina platensis, a blue - green algae, is unicellular and has cylindrical cells in un-branched
helicoidal trichomes measuring 6.00-8.00µm in diameter. The name “Spirulina” is derived
from the Latin word “Helix” or “Spiral”. For the first time, Spirulina maxima was found in
lake Texcoco in Mexico. Spirulina belongs to Phylum: Cyanobacteria, Class:
Cyanobacteriaceae, Order: Nostocales, Family: Oscillatoriaceae, Genus: Spirulina.
The term “Spirulina” includes various species of primitive unicellular blue-green algae,
most commonly Spirulina maxima and Spirulina platensis. These microscopic plants grow
naturally in lakes rich in salt.
Spirulina is a ubiquitous organism. After the first isolation by Turpin in 1827 from a
freshwater stream, several species of Spirulina have been found in a variety of environments
i.e. soil, sand, marshes, brackish water, seawater and freshwater. Different species of Spirulina
have been isolated from different sources like tropical water, thermal springs, warm water from
power plants and fresh ponds. Thus the organism appears to be capable of adaptation to vary
different habitats and colonizes certain environment in which life for other microorganisms
becomes difficult (Mueller et al., 1975).
Spirulina has been labeled as the ideal foodstuff and recommended by United Nations
Food Agriculture Organization (UNFAO) due to its highly nutritious quality. The nutritive
value of these algae is extremely high. Protein content of one kg of mixed vegetables with
Spirulina is as high as 70 per cent and is five times higher compare to the chicken egg. It is
regarded as one of the richest sources of proteins in the world. In addition, Spirulina is also
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rich in amino acids, vitamins, sugars and trace elements. Hence, it is popularly used as a
natural immunomodulator and rejuvenator in humans (specifically infants and women),
animals and poultry. The chemical composition of Spirulina platensis as reported by Balloni
et al. (1980) is as follows:
Chemical composition Percentage
Protein 51-71
Carbohydrate 14
Lipids 6
The amino acid composition of Spirulina platensis, as reported by Chronakis, (2001) is
presented hereunder:
Amino Acids (essentials)
Spirulina grams
Egg Protein per 100grams
FAO Standard proteins
Isoleucine 6.40 5.80 4.00
Leucine 10.40 9.00 7.00
Lysine 4.50 6.70 55.00
Methionine + Cystine 2.20 3.00 55.00
Phenylalanine 5.40 5.30 6.00
Threonine 5.40 5.30 4.00
Tryptophane 1.50 1.80 1.00 Valine 7.50 7.20 5.00
The carbohydrates composition of Spirulina platensis, as reported by Pugh et al. (2001)
is presented hereunder:
Carbohydrate contents Amount (grams per 100 grams) Ramanose 9.00
Glucan 1.50
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Phosphorylated Cyclitols 2.50
Glucosamine Muramic acid 2.00
Glycogen 0.50
Sialic acid and others 0.50
The lipid profile of Spirulina platensis, as reported by Cohen (1997) is presented
hereunder:
Lipids content (principal ones) Amount (mg/kg) Palmitic acid (saturated fatty acid) 16,500 to 21,141
Linoleic acid (unsaturated FA) 10,920 to 13,784
Gamma linoleic acid (omega 6) 8,750 to 11,970
Alpha linolenic acid (omega 3) 699 to 7,000
Chlorophyll-a 6,100 to 7,600
Beta sitosterol 30 to 97
Beta carotene average 1,700
The composition of vitamins in Spirulina platensis, as reported by Belay (1997) is
presented hereunder:
Vitamins Amount (mg/kg) Biotin 0.40
Cyanocobalamin ( B12 ) 0.45
Delta-calcium Panthothenate 11.00
Folic acid 0.50
Inositol 350.00
Nicotinic acid ( PP ) 118.00
Pyridoxine ( B6 ) 3.00
Riboflavine ( B2 ) 40.00
Thiamin ( B1 ) 55.00
Tocopherol ( E ) 190.00
Ascorbic acid ( C ) 90.00
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Due to the fact that Spirulina is a whole organism, it contains many important nutrients,
including all of the essential amino acids, vitamins, minerals, and essential fatty acids. Feeding,
Spirulina as a source of protein, vitamin and mineral supplement in poultry was stated as early
as 1990’s (Ross and Dominy, 1990).
Ross et al. (1994) reported that Spirulina improved the growth performance and
immune performance of chickens, while Venkataraman et al. (1994) used it as a source of
carotenoids for egg yolk and broiler pigmentation.
Exploration of literature revealed that the research is scanty on the utility of Spirulina
in countering aflatoxicosis, particularly in chicken. Raju et al. (2004) reported improved body
weight, dressing yields and cellular immune response by Spirulina in the broiler diet at 0.02
% level, while no such effect on feed intake, organ weights and humoral immune response in
broiler chicken fed 300ppb AFB1.
Verma and Nair (2004) reported that Spirulina inclusion in broiler diet at 1 per cent
showed profound antioxidant effects in terms of increased activity of erythrocyte antioxidant
enzymes and decreased serum lipid peroxidation.
Spirulina is also a rich source of gamma linoleic acid (GLA), an essential fatty acid
found in food as well as in herbal extracts such as evening primrose oil. Spirulina also contains
a novel polysaccharide called Calcium spirulan (Ca-SP) that may have antiviral and anti-
thrombic activity (Mueller et al., 1975).
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2.6.3. Use of enzymes
The enzymes are believed to break the functional atomic group of the mycotoxin
molecule and thereby render them nontoxic (Kumar et al., 1993). Enzymes viz.,
carboxyesterase present in the microsomal fraction of the liver, esterase and epoxidase are
being tried for their practical applicability in the field conditions (Pasteiner, 1997).
2.6.4. Nutritional manipulations
Increasing the crude protein content and supplementation of additional levels of
riboflavin, pyridoxine, folic acid and choline showed protective effect against aflatoxicosis
(Ehrich et al., 1986). Anti-oxidants like BHT and -napthoflavone, vitamin C and vitamin E
offer protection against AF induced genotoxicity in in vitro studies (Johri et al., 1990).
Increase in dietary protein levels and supplementation of L-phenylalanine was revealed
to be effective against aflatoxicosis and ochratoxicosis. Devegowda et al. (1998) reported that
the supplementation of the diet with selenium and methionine partially alleviated the adverse
effects of AF respectively.
2.6.5. Biological methods
With the awareness of potential harmful effects of chemicals used for counteracting
mycotoxins and the cost involvement with their usage has prompted the scientists to look for
alternative methods which are applicable and safe. A rapid explosion in the field of feed
industry through the biotechnological methods has opened a new possibility of degradation of
mycotoxins by microorganisms. Several yeasts, moulds and bacterial strains posses the ability
either to destroy or transform mycotoxins successfully (Phillips et al., 1988).
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2.6.5.1. Bacterial degradation
Acid producing bacteria’s such as Lactobacillus plantarum and Lactobacillus
acidophilus were found to detoxify AF in maize. Rumen bacteria were found to degrade
ochratoxin A (OA), T-2 toxin and zearalenone (ZEA) (Linderfelser and Ceigler, 1970).
He et al. (1992) detoxified moldy maize diet containing 5.00ppm vomitoxin,
microbially through incubation with the contents of large intestine of chickens having a
detoxified vomitoxin of 2.10ppm.
2.6.5.2. Protozoan degradation
Tetrahymena pyriformis at a dose rate of 22x106 cells detoxified AFB1, by converting it
into its hydroxyl products to an extent of 5 per cent in 24 hours and 67 per cent in 48 hours
(Robertson et al., 1970). Intact rumen fluid containing various protozoa was reported to
metabolize T-2 toxin and ochratoxin while no effect on AF was noted (Kiessling et al., 1984).
2.6.5.3. Fungal degradation
Some of the species of fungi have been found to detoxify AF. An intracellular
substance was found to be responsible for A. flavus and A. parasiticus to degrade the formed
toxins in a culture when their mycelium was subjected to fragmentation. The peroxidase
enzymes produced by the fungal mycelium, which can catalyze hydrogen peroxide into free
radicals, reacts with AF (Dvorak, 1989).
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2.6.5.4. Degradation by yeast
Yeasts are being primarily used as growth promoters in poultry and animal feeds.
Besides their beneficial effects on feed utilization and rich concentration of many vitamins,
certain species and strains of yeasts have been observed to detoxify mycotoxins through its
degradation (Cooney, 1980).
Supplementation of live cells of Saccharmyces cerevisiae1026 was found to be beneficial
in counteracting the adverse effect of several mycotoxins (Stanley et al., 1993). It has also
improved serum total protein and HI titer against Newcastle disease in AF fed broilers
(Devegowda et al., 1996). In an another study, inactivated yeast at 0.2 per cent in the diet was
found to alleviate the growth depression effects of AF up to 200ppb level (Devegowda et al.,
1998).
Mycotoxin binding ability of Maannanoligosaccharides has been demonstrated in
various in vitro trials (Devegowda et al., 1998) and in vivo trials (Raju and Devegowda, 2000;
Swamy et al., 2004).
2.6.5.5. Counteraction of aflatoxin by Mannanoligosaccharide
Until late 1960s, carbohydrates were thought to serve only as energy source (in the
form of monosaccharides and storage molecules such as the poly saccharine starch) and as
structural materials (the polysaccharides cellulose in plants and chitin in the exoskeletons of
insects) and also forming an integral part of structural elements such as nucleic acids,
glycolipids and glycoproteins. However, biotechnologists have explored the role it plays in
66
various other biological functions especially in enhancing the immune response and their
ability to block colonization and to bind various pathogens (Girish et al., 2008).
2.6.5.5.1. Oligosaccharide
Oligosaccharides include a wide range of molecules which are natural constituents of
plants and microorganisms. They are complex carbohydrates composed of at least three
carbohydrate monomers which comprised of similar or different building blocks, different
linkages between components and may be linear or branched. With the exception of malto-
dextrins derived from starch, most oligosaccharides have a composition that cannot be
degraded by digestive enzymes of mammals or birds. This allows complex carbohydrates to
avoid enzymatic digestion by the host and they serve as nutrients for the gastrointestinal
microflora particularly for the beneficial type of microbes (Terada et al., 1994).
Some of the complex sugars, such as raffinose or stachyose are abundant in beans, peas
and soybeans and have anti nutritive value for the animals. Other such as fructooligosaccharide
(FOS) galatcooligosaccharides (GOS) and lactosucrose (LS) and mannanoligosaccharides
(MOS) have shown the potential to improve health and performance when added to animal
diets (Savage et al., 1996). Paterson et al. (1997) stated that MOS comprises of chains of
mannose sugar, the back bone of this chain are α-1, 6 with side chains bound by α-1, 2 and α-1,
3 linkages. MOS have been investigated for their impact on gut microbiology with the goal of
lessening the impact of pathogen’s challenge to the young animals or birds.
FOS consists primarily of one to three fructose residues attached to a sucrose molecule
and are found naturally in relatively high concentrations in onions, wheat, barley and rye. FOS
67
is selectively used by beneficial bacteria such as Bifidobacteria. Effect of FOS on broiler
growth rate, feed utilization and mortality has been inconsistent (Yegani et al., 2006).
2.6.5.5.2. Mannanoligosaccharides derived from cell wall
MOS are prepared from the yeast cell wall of Saccharomyces cervisiae. Glucan,
mannan and chitin are the main components of yeast cell wall. The basic composition of the
wall consists of 46 per cent glucan, 43 per cent mannan, 12.5 per cent protein, 1.1 per cent
hexosamines, 0.79 per cent non hexosamine nitrogen and 0.4 per cent phosphorus (Mill, 1966),
while the ratio of one component to another remains relatively constant from strain to strain.
The degree of mannan phosphorylation and interaction between mannan, glucan and protein
components vary. Glucan is thought to make up the matrix of the cell wall which is covered by
another layer of mannose sugar. These mannose sugars are arranged in a chain of
mannopyranoside residues. The linkages in the back bone of this chain are α-1, 2 and α-1, 3
linkages and posses powerful antigen stimulating properties. As the mannan extracted from the
yeast cell wall is remarkably stable to acid digestion, it can survive while passing through the
stomach or abomasum and remain undisturbed, and that may account for the product biological
activity in such a wide range of species. Yeast cultures have been traditionally used as growth
promoters in monogasteric animals (Bradley et al., 1994) and as mycotoxin counteracting
agents (Girish et al., 2008).
2.6.5.5.3. Mode of action of Mannanoligosaccharide
The exact mechanism by which modified - MOS brings about its beneficial effect in
animal is not entirely understood. However, possible modes of action have been proposed.
68
These include blocking the colonization of pathogens, stimulation of immune response,
provision of nutrients that cannot be used by pathogens and mycotoxin absorption.
2.6.5.5.3.1. Blocking of colonization by pathogens
The role of carbohydrates on the surface of cells and their function in cell to cell
interactions has been described by Sharon and Lis (1993). A number of specific interactions
involving adherence of bacteria to the mucosal lining of the intestinal tract exists. Many of the
key sugars involved in these reactions contain mannose. These sugar residues serve as
receptors on the surface of cells in the intestinal wall and interact with specific proteins of the
bacterial cells called lectins.
Mannose specific lectins allow a number of specific bacteria to attach to the intestinal
wall and provide anchors against the flow of ingesta and mucus which act to continually flush
microorganisms from the intestinal tract. These attachments provide a mechanism, which
allows slow growing organisms to colonize the inner surface of the intestinal tract. Binding of
Salmonella, Escherichia coli and Vibro cholera has been shown to be mediated by a mannose
specific lectin like substance on the bacterial cell surface.
MOS added to feeds, provide the mannose sugar that can attach to the bacterial lectins
and prevent the colonization in the host intestine. Besides, modified-MOS has also been known
to bind to specific receptor sites of the GI tract epithelium and prevent bacterial colonization by
competitive exclusion (Newman, 1995). Salmonella colonization reduced from 76 per cent to
18 per cent by dietary modified-MOS inclusion. A commercial turkey flock supplemented with
modified-MOS at 2lb/ton of feed for three weeks followed by 1lb/ton of feed upto 17 weeks
had a better overall performance, decreased incidence of enteric diseases and high antibody
69
titers to haemorrhagic enteritis and Newcastle Disease as compared to respective control
(Olsen, 1995).
2.6.5.5.3.2. Stimulation of immune response
It is known that specific polysaccharides of microbial origin act as adjuvants
(enhancing agent) when added to various types of vaccines. The presence of the appropriate
adjuvant dramatically improves antibody response and thus the degree of protection provided
by the vaccine. Further, these polysaccharides have been reported to act as antigenic substances
themselves, eliciting direct antibody response.
Mannose sugars in the MOS influenced the immune system by causing the animal to
release interleukin–6 which inturn stimulates the secretion of mannose binding protein from
the liver which binds to the capsule of invading bacteria and triggers off the complement
fixation system. McDonald (1995) established that modified-MOS stimulated the immune
response by increasing the phagocytic activity of macrophages incubated with the peripheral
blood of wistar rats. They also observed the response of broiler chicks challenged with wild
strains of salmonella and found that birds provided modified-MOS at the rate of 1g/kg in the
diet were able to withstand the challenge better. Newman (1995) also established the
adsorption property of modified-MOS to several strains of clostridia and salmonella in an in
vitro study.
In turkeys, increased concentrations of plasma IgG (26%), bile IgA (30%), plasma
sodium and albumin levels and lowered activity of gamma glutamyl transferase (GGT) were
observed by feeding modified-MOS at the rate of 0.1 per cent in the diet (Savage et al., 1996).
The exact mechanism involved in immune stimulation is still not fully understood. However, it
70
is assumed that a small portion of modified-MOS might be taken up in the small intestine by
M-cells, causing B-cell activation and subsequent activation of T-cells and macrophages.
When modified-MOS was included in the diets of growing pullets, there was a
modulation of a standard measure of a cell mediated immune function, the phytohemaglutin
(PHA) wattle reaction (Cotter and Weinner, 1997).
2.6.5.5.3.3. Mycotoxin adsorption
Pathogenic bacteria are unable to utilize the sugars present in modified-MOS as a
source of energy for growth. Beneficial bacteria on the other hand, seem to be stimulated in the
presence of modified-MOS. Addition of 0.1 per cent modified-MOS at the level of 0.05 per
cent significantly improved live weight and FCR in broiler birds. When modified-MOS was
incorporated to the diet of White Leghorn hens at 0.05 per cent, there was 10 per cent increase
in egg production (Chuckwu and Stanley, 1997).
Mannanoligosaccharide derived from the cell wall of Saccharomyes cerevisiae appears
to have high affinity for a wide range of mycotoxins. Although the exact mechanism is not
known, it is hypothesized that the glucomannan matrix of commercial Glucomannan
Mycotoxin Adsorbent (GMA) preparation traps the mycotoxins in an irreversible way (Afzali,
1998).
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2.6.5.5.3.3.1. Glucomannan - Extract Binder:
The advent of biotechnology in the last decade opened a new avenue for tackling the
problem of mycotoxicosis by using yeast extracts. Yeasts are being primarily used as growth
promoters in poultry and animal feeds (Day et al., 1987). Supplementation of live cells of
Saccharmyces cerevisiae1026 was found to be beneficial in counteracting in adverse effect of
several mycotoxins (Stanley et al., 1993).
At the lowest levels of inclusion also this yeast product bound to AF in poultry feed and
at highest levels of inclusion, modified glucomannan bound to AF at about 70 per cent.
Glucomannan - Extract Binder also improved serum total protein and HI titer against
Newcastle Disease in AF fed broilers (Devegowda et al., 1996). In vitro study conducted by
Mahesh and Devegowda (1996) showed the AF binding ability of modified glucomannan in
contaminated poultry feeds.
Channakrishnappa, (1997) observed that inactivated yeast at 0.2 per cent in the diet
alleviated the growth depression effects of AF up to 200ppb level.
In a series of in vitro experiments, Afzali (1998) reported that GMA was found to bind
zearalenone to the extent of 65 per cent and was pH dependent.
Trial by Afzali and Devegowda (1999) showed that modified glucomannan
supplementation (0.1% and 0.2%) resulted in improved egg production and immune response
of broiler breeders fed with graded levels of AF (50, 100 and 200ppb).
72
Yegani et al. (2006) reported that the feeding of mycotoxin contaminated grains
decreased eggshell thickness. Dietary supplementation with Glucomannan Mycotoxin
Adsorbent (GMA) prevented this effect. There was a significant increase in early (1 to 7 d)
embryonic mortality in eggs from birds fed mycotoxin contaminated grains. The feeding of
mycotoxin contaminated grains decreased antibody titers against infectious bronchitis virus
and this was prevented by dietary supplementation with GMA.