19

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

20

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,

21

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

22

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).

23

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

24

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

25

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.

26

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

27

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

28

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

29

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.

30

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.

31

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.

32

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.

33

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

34

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.

35

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.

36

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

37

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.

38

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

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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

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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.

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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).

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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.