Ninety-six laying hens fed with 2.50ppm of aflatoxin B1 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 aflatoxin influenced color parameters, which was attributed to interference of aflatoxin B1 with lipid metabolism and pigmentary substances deposition in yolk. Further, no aflatoxin B1 or aflatoxin M1 residues were found in eggs of the experimental groups.

Pandey & Chauhan (2007) reported that feeding of aflatoxin B1 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 aflatoxin B1 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

1.25, 2.50, 5 and 10ppm aflatoxin B1 in layer rations for four weeks and found decreased egg

Fernandez et al. (1994) reported a significant reduction in egg production and oral lesions in

Azzam & Gabal, (1998) reported a significant reduction in egg production of commercial

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 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

Mukhopadhy et al. (2000) have also reported a significant reduction in egg production in

Ginzberg et al. (2000) reported that the yolk color in the group fed on 5 per cent of *Spirulina*

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

Kim et al. (2003) found reduction in serum calcium, phosphorous and ALT and increase in gamma glutamyl transferase (GGT) levels in laying hens by dietary levels of 500ppb of

Chowdhury & Smith (2004) reported decrease in feed efficiency when layers fed *Fusarium*

Ogido et al. (2004) reported an increase in feed consumption and decrease in egg weight in Japanese quails fed with combination of 50ppb of aflatoxin B1 and 10ppm of fumonisin B1

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

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 aflatoxin B1 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 aflatoxin influenced color parameters, which was attributed to interference of aflatoxin B1 with lipid metabolism and pigmentary substances deposition in yolk. Further, no aflatoxin B1 or aflatoxin M1 residues were found in eggs of the

Pandey & Chauhan (2007) reported that feeding of aflatoxin B1 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 aflatoxin B1 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

production in birds receiving 5 and 10ppm of aflatoxin B1.

layers fed with high levels of aflatoxin for six weeks.

with 100mg toxin/kg diet.

for 140d.

experimental groups.

layer chicken treated with 120ppb onwards for varying periods.

commercial layers exposed to 500ppb aflatoxin given for 90 days.

algae was 2.4 times darker compared to the control laying hens.

mycotoxins contaminated diets compared with control groups.

aflatoxinB1 for 42 d to White Leghorn hens aged 42 weeks.

increase significantly with the dosage of *Spirulina*.

aflatoxin given from week 67 in laying hens.

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.

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.

#### **6. Aflatoxicosis in breeders**

When aflatoxin (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 & Wyatt (1976) fed broiler breeder hens 5 and 10ppm of aflatoxin 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 aflatoxin fed groups. Egg production decreased significantly during weeks three and four after initiation of toxin feeding in hens fed with 10 and 5ppm aflatoxin, respectively. They also observed enlarged fatty and friable liver and enlarged spleens by feeding aflatoxin at the dose levels of 0.00, 5.00 and 10.00ppm. Further, they did not observe any latent effect of aflatoxin 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 aflatoxin for four weeks.

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 aflatoxin 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 aflatoxin B1 or 1.60ppm aflatoxin G1 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).

Jayakumar et al. (1988) fed aflatoxin B1 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 aflatoxin B1 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 aflatoxin containing eggs and concluded that it was low in comparison to chicks hatched from aflatoxin free eggs. Further, they studied the post-hatch performance of chicks hatched from aflatoxin containing eggs and observed lower weight gains and impaired defense system in chicks fed on normal diet.

In a study by Abdelhamid & Dorra (1990) where the maternal diet contained 100.00ppb of aflatoxin, citrinin or patulin for six weeks, the chicks had significantly higher weight than the control.

Aflatoxicosis in Layer and Breeder Hens 211

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

Histological evidence of adverse effects of aflatoxin on the germinal epithelium of the testes was reported in immature chickens dosed with 200µg of aflatoxin/day/chick for 35 days

Manafi et al. (2009) reported the aflatoxin fed at the levels of 300, 400 and 500ppb for three periods, each with duration of three weeks to broiler breeders from 28 to 36 weeks of age. Inclusion of 500ppb aflatoxin in the diet significantly (P≤0.05) affected feed consumption, feed efficiency, egg production, egg weight, fertility, hatchability, embryonic mortality, GGT and ALT levels in the serum, organ lesion scores (liver, kidney, proventriculus and gizzard), relative weights of heart as well as liver, antibody titers against Newcastle and Infectious bursal diseases when compared to that of control. The results indicated no significant (P≥0.05) effect of aflatoxin on body weight, shell thickness, Haugh unit, residue in eggs, sperm count, per cent live sperm, yolk color index and relative weight of spleen when

Serum alkaline phosphatase levels were significantly higher, serum alkaline aminotransferase (*P*=0.068) and gamma-glutamyltransferase (*P*=0.067) levels tended to increase (*P*<0.05) in 58-wk-old Ross 308 broiler breeders fed with 100 µg aflatoxincontaminated diet than those of hens fed the uncontaminated diet (Matur et al., 2010).

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 aflatoxin B1 and M1, (aflatoxin M1 is a polar metabolite of aflatoxin B1) and ochratoxin A, when present in animal products are a threat to human health and must be monitored. The limit for aflatoxin B1 in complete feeds

Trucsksess et al. (1983) were able to detect aflatoxin B1 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 aflatoxin occurs faster from the albumen than

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

Zaghini et al. (2005) reported that no traces of aflatoxin B1 or aflatoxin M1 residues were

In another study, Salwa & Anwer (2009) reported no traces of aflatoxin in the eggs of layers

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

residual aflatoxin B1 or its metabolites were found in eggs (Oliveira et al., 2002).

fed with 25.00, 50.00 and 100ppb of aflatoxin in their diet for 60 days.

found in eggs of layer hens supplemented with diet containing 2.50ppm aflatoxin B1.

on the relative weights of liver, spleen, kidney and testes.

(Mohan et al., 2008).

compared to that of control.

**7. Aflatoxin residue in eggs**

**8. Counteraction of aflatoxicosis**

is 0.02mg/kg.

from the yolk.

Rao (1990) observed a drastic deterioration in semen quality of breeder cocks fed with 1.000ppm aflatoxin. 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 aflatoxin for three weeks.

Nelson-oritiz & Qureshi (1992) assessed single dose exposure of six day-old embryos to 0.100, 0.500 and 1.00µl of aflatoxin B1 and concluded that rate of mortality of the embryos was dose related. Chick embryos, administered different levels of aflatoxin or ochratoxin on the chorio-allontoic membrane showed decreased weight and length.

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 & Sugahara (1995) reported that birds fed aflatoxin 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 aflatoxin B1 (0.00, 0.50, 1.00 and 1.50ppm) on the reproductive performance of layer breeders. He reported that the sperm motility and concentration were not affected while the percentage of sperm abnormality increased when aflatoxin B1 was included in the diet of breeder cocks. The feed consumption was significantly decreased and egg production declined in proportion to the level of aflatoxin B1 incorporation in the diets. There was no effect on fertility but hatchability was affected. The chicks hatched from breeder hens, received graded levels of aflatoxin 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 & Weinner (1997) reported lowered hatchability in broiler breeder hens fed with four levels viz., 0.00, 308.00, 610.00 and 1834.00ppb of aflatoxin.

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, they have 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.

Stanley et al. (2004) reported that feeding aflatoxin 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 (1 to 7d) 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

Rao (1990) observed a drastic deterioration in semen quality of breeder cocks fed with 1.000ppm aflatoxin. The traits affected were semen volume, semen concentration, motility

Stephen et al. (1991) reported a significant drop in egg production in layer chicken fed with

Nelson-oritiz & Qureshi (1992) assessed single dose exposure of six day-old embryos to 0.100, 0.500 and 1.00µl of aflatoxin B1 and concluded that rate of mortality of the embryos was dose related. Chick embryos, administered different levels of aflatoxin 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

Bergsjo et al. (1993) reported chick developmental anomalies when laying hens were fed

Diaz & Sugahara (1995) reported that birds fed aflatoxin at 0.66 or 3.00µg/kg diet did not

Muthiah (1996) conducted an experiment to study the effect of graded dietary levels of aflatoxin B1 (0.00, 0.50, 1.00 and 1.50ppm) on the reproductive performance of layer breeders. He reported that the sperm motility and concentration were not affected while the percentage of sperm abnormality increased when aflatoxin B1 was included in the diet of breeder cocks. The feed consumption was significantly decreased and egg production declined in proportion to the level of aflatoxin B1 incorporation in the diets. There was no effect on fertility but hatchability was affected. The chicks hatched from breeder hens, received graded levels of aflatoxin 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

Cotter & Weinner (1997) reported lowered hatchability in broiler breeder hens fed with four

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, they have demonstrated that feeding diets contaminated with 10.00 and 20.00mg of DON per kg of feed decreased the fertility in broiler breeder males,

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

Stanley et al. (2004) reported that feeding aflatoxin at the rate of 3mg/kg to 35 week's old Cobb broiler breeder hens for three weeks significantly reduced serum total protein,

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

Yegani et al. (2006) reported no effect in feed consumption, body weight, and egg production. However, increase in early embryonic mortality (1 to 7d) 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

and abnormalities.

period.

wk of age.

to embryonic mortality.

twisted limbs were observed.

5.00 and 10.00ppm aflatoxin for three weeks.

diets containing 4.90mg of DON/kg of feed for 10 weeks.

levels viz., 0.00, 308.00, 610.00 and 1834.00ppb of aflatoxin.

albumin, calcium and phosphorus levels.

though there was no difference in the volume of semen produced.

show any adverse effect on chick performance.

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.

Histological evidence of adverse effects of aflatoxin on the germinal epithelium of the testes was reported in immature chickens dosed with 200µg of aflatoxin/day/chick for 35 days (Mohan et al., 2008).

Manafi et al. (2009) reported the aflatoxin fed at the levels of 300, 400 and 500ppb for three periods, each with duration of three weeks to broiler breeders from 28 to 36 weeks of age. Inclusion of 500ppb aflatoxin in the diet significantly (P≤0.05) affected feed consumption, feed efficiency, egg production, egg weight, fertility, hatchability, embryonic mortality, GGT and ALT levels in the serum, organ lesion scores (liver, kidney, proventriculus and gizzard), relative weights of heart as well as liver, antibody titers against Newcastle and Infectious bursal diseases when compared to that of control. The results indicated no significant (P≥0.05) effect of aflatoxin on body weight, shell thickness, Haugh unit, residue in eggs, sperm count, per cent live sperm, yolk color index and relative weight of spleen when compared to that of control.

Serum alkaline phosphatase levels were significantly higher, serum alkaline aminotransferase (*P*=0.068) and gamma-glutamyltransferase (*P*=0.067) levels tended to increase (*P*<0.05) in 58-wk-old Ross 308 broiler breeders fed with 100 µg aflatoxincontaminated diet than those of hens fed the uncontaminated diet (Matur et al., 2010).

#### **7. 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 aflatoxin B1 and M1, (aflatoxin M1 is a polar metabolite of aflatoxin B1) and ochratoxin A, when present in animal products are a threat to human health and must be monitored. The limit for aflatoxin B1 in complete feeds is 0.02mg/kg.

Trucsksess et al. (1983) were able to detect aflatoxin B1 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 aflatoxin 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 aflatoxin B1 or its metabolites were found in eggs (Oliveira et al., 2002).

Zaghini et al. (2005) reported that no traces of aflatoxin B1 or aflatoxin M1 residues were found in eggs of layer hens supplemented with diet containing 2.50ppm aflatoxin B1.

In another study, Salwa & Anwer (2009) reported no traces of aflatoxin in the eggs of layers fed with 25.00, 50.00 and 100ppb of aflatoxin in their diet for 60 days.

#### **8. 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

Aflatoxicosis in Layer and Breeder Hens 213

basil, and mint) reduced free radical oxygen formation, and inhibited protein/DNA synthesis as well as apoptosis of human hepatoma cells caused by aflatoxin B1 and

According to Gowda & Ledoux (2008) ellagic acid, a phenolic compound of strawberries and grapes showed anticarcinogenic activity and inhibited aflatoxin B1 mutagenicity. Smethyl methane thiosulfonate present in cabbage and onion suppressed chromosomal aberrations due to aflatoxin B1 in rat bone marrow cells. Diterpenes, cafestol and kahweol present in green and roasted coffee beans prevented the covalent binding of aflatoxin B1 to

Iqbal et al. (1983) observed chemoprotective effect of piperine (1-piperoyl piperidine), an alkaloid of pepper against aflatoxin by inhibiting cytochrome P 450 bioactivation of aflatoxin B1. The protective effect of chlorophylline (a derivative of the green pigment chlorophyll) against aflatoxin B1 was also observed. The toxic effects of aflatoxin 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

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

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 aflatoxin 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

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 aflatoxin 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

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

ochratoxin A (Manafi et al., 2009).

anion generation was noticed.

**8.4 Nutritional manipulations**

adverse effects of aflatoxin respectively.

adverse effects of aflatoxin respectively.

**8.6 Biological methods**

**8.5 Nutritional manipulations**

**8.3 Use of enzymes**

DNA by modulation of the carcinogenesis enzyme system.

their practical applicability in the field conditions (Pasteiner, 1997).

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:

#### **8.1 Physical methods**

According to Park & Liang (1993) Mycotoxins (aflatoxin, 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 (Manafi et al., 2009).

Clays like hydrated sodium calcium aluminosilicates, activated carbon, bentonite, clays and special polymers 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 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.

#### **8.2 Herbal methods**

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.

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 aflatoxin B1. Feeding of the extract of *Azadirachta indica* prevented metabolic activation of aflatoxin B1 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 aflatoxin B1.

Oxidative changes (increased peroxides, reduced antioxidant enzyme activity) in liver and kidney due to aflatoxin B1 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,

procedures and products to effectively deal with the decontamination and remediation of mycotoxin contamination in feedstuffs. The available approaches were reviewed by

The methods aimed at preventing or reducing the level of mycotoxin contamination were

According to Park & Liang (1993) Mycotoxins (aflatoxin, ochratoxin, T-2 toxin and citrinin) are highly soluble in organic solvents. Their extraction from the feed stuffs using several

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

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

Clays like hydrated sodium calcium aluminosilicates, activated carbon, bentonite, clays and special polymers 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 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

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

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 aflatoxin B1. Feeding of the extract of *Azadirachta indica* prevented metabolic activation of aflatoxin B1 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

Oxidative changes (increased peroxides, reduced antioxidant enzyme activity) in liver and kidney due to aflatoxin B1 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,

classified as preventive or curative. The following approaches were recommended: 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.

Trenholm et al. (1996) and Devegowda et al. (2003).

4. Utilization of mycotoxin resistant genetic resources.

effective. These methods have little practical applicability.

essential nutrients from the diet (Manafi et al., 2009).

animals and poultry through their antioxidant activity.

(cations) that are components of the layers.

**8.2 Herbal methods**

protection against aflatoxin B1.

solvents or mixture of solvents has been proved to be highly effective.

**8.1 Physical methods**

basil, and mint) reduced free radical oxygen formation, and inhibited protein/DNA synthesis as well as apoptosis of human hepatoma cells caused by aflatoxin B1 and ochratoxin A (Manafi et al., 2009).

According to Gowda & Ledoux (2008) ellagic acid, a phenolic compound of strawberries and grapes showed anticarcinogenic activity and inhibited aflatoxin B1 mutagenicity. Smethyl methane thiosulfonate present in cabbage and onion suppressed chromosomal aberrations due to aflatoxin B1 in rat bone marrow cells. Diterpenes, cafestol and kahweol present in green and roasted coffee beans prevented the covalent binding of aflatoxin B1 to DNA by modulation of the carcinogenesis enzyme system.

Iqbal et al. (1983) observed chemoprotective effect of piperine (1-piperoyl piperidine), an alkaloid of pepper against aflatoxin by inhibiting cytochrome P 450 bioactivation of aflatoxin B1. The protective effect of chlorophylline (a derivative of the green pigment chlorophyll) against aflatoxin B1 was also observed. The toxic effects of aflatoxin 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.

#### **8.3 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).

#### **8.4 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 aflatoxin 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 aflatoxin respectively.

#### **8.5 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 aflatoxin 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 aflatoxin respectively.

#### **8.6 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

Aflatoxicosis in Layer and Breeder Hens 215

The addition of aflatoxin in layer and breeder hens could lead to aflatoxicosis which can adversely affect the performance and reproductive efficacy of the birds. 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 up to millions of dollars. In commercial Layers, the most prominent manifestations of experimental aflatoxicosis are reduced egg production and egg weight, increased liver fat and alterations in some serum biochemical parameters.In case of breeder hens, many of parameters like changes in body weight, feed consumption, feed conversion ratio, egg production, egg weight, shell thickness, Haugh unit score, yolk color index, fertility, hatchability and embryonic mortality and some of biochemical and immunological parameters like serum levels of GGT and ALT, visceral organ weight and organ lesions and serum antibody titers for ND and IBD could be altered due to aflatoxicosis. This chapter briefly reviews the impact of aflatoxicosis in

Abdelhamid, A.M. & Dorra, T.M. (1990). Study on effects of feeding laying hens on separate

Azzam, A.H. & Gabal, M.A. (1998). Aflatoxin and immunity in layer hens. *Avian Path.,* 27:

Bergsjo, B., Herstad, O. & Nafstad, I. (1993). Effects of feeding deoxynivalenol-contaminated

boulton, S.L., dick, J.W. & hughes, B.L. (1981). Effects of dietary aflatoxin and ammonia –

Brake, J., Hamilton, P.B. & Kittrell, R.S. (1999). Effect of the trichothecene mycotoxin

Brake, J., Hamilton, P.B. & Kittrell. (2000). Effects of the trichothecene mycotoxin

Briggs, D.M., Wyatt, R.D. & Hamilton, P.B. (1974). The effect of dietary aflatoxin on semen characteristics of mature broiler breeder males. *Poult. Sci.,* 53: 2115-2119. Chandrashekharan, D. (2000). Mycotoxins: A threat to livestock and public health, Personal

Chandrashekharan, D., Sudaram, T.K. & Natrajan, A. (2002). Mycotoxin scenario in India and control strategies. IV Biennial Conference and Exhibition, Kolkata, pp. 34-41. Chowdhury, S.R. & SMITH, T.K., 2004. Effects of feeding blends of grains naturally

Cooney, D.D. (1980). Activated charcoal: antidotal and other medical uses. Marcel Dekker

and tissue constituents. *Arch. Tierernahr.,* 40: 305-315.

mycotoxins (aflatoxin, patulin, or citrinin) contaminated diets on the egg quality

oats on reproductive performance in White Leghorn hens. *Br. Poult. Sci.,* 34: 147–

inactivated aflatoxin on Newcastle disease antibody titers in layer – breeders. *Avian*

diacetoxyscirpenol on fertility and hatchability of broiler breeders. *Poult. Sci.,* 78:

diacetoxyscirpenol on feed consumption, body weight and oral lesions of broiler

contaminated with *Fusarium* mycotoxins on performance and metabolism of laying

**9. Conclusion**

commercial layer and breeder hens.

**10. References**

570-577.

*Dis.,* 26: 1-6.

1690–1694.

Communication.

breeders. *Poult. Sci.,* 79; 856-863.

hens. *Poult. Sci.,* 83: 1849–1856.

Inc., New York, USA.

159.

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).

#### **8.6.1 Bacterial degradation**

Acid producing bacteria's such as *Lactobacillus plantarum* and *Lactobacillus acidophilus* were found to detoxify aflatoxin in maize. Rumen bacteria were found to degrade ochratoxin A (OA), T-2 toxin and zearalenone (ZEA) (Linderfelser & 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.

#### **8.6.2 Protozoan degradation**

*Tetrahymena pyriformis* at a dose rate of 22x106 cells detoxified aflatoxin B1, 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 aflatoxin was noted (Kiessling et al., 1984).

#### **8.6.3 Fungal degradation**

Some of the species of fungi have been found to detoxify aflatoxin. 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 aflatoxin (Dvorak, 1989).

#### **8.6.4 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).

In the early 1990s, a commercially yeast culture preparation of *Saccharomyces cerevisiae1026*, which was earlier noted as digestive aid and a growth promoter, was found to improve hatchability (McDaniel, 1991) and broiler body weights (Stanley et al., 1993). Further investigations lead to the establishment of yeast culture preparation's ability to adsorb aflatoxins in poultry feeds (Devegowda et al., 1995).

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 aflatoxin 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 aflatoxin 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 & Devegowda, 2000; Swamy et al., 2004).

#### **9. Conclusion**

214 Aflatoxins – Biochemistry and Molecular Biology

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

Acid producing bacteria's such as *Lactobacillus plantarum* and *Lactobacillus acidophilus* were found to detoxify aflatoxin in maize. Rumen bacteria were found to degrade ochratoxin A

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

*Tetrahymena pyriformis* at a dose rate of 22x106 cells detoxified aflatoxin B1, 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 aflatoxin was noted (Kiessling et al.,

Some of the species of fungi have been found to detoxify aflatoxin. 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

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

In the early 1990s, a commercially yeast culture preparation of *Saccharomyces cerevisiae1026*, which was earlier noted as digestive aid and a growth promoter, was found to improve hatchability (McDaniel, 1991) and broiler body weights (Stanley et al., 1993). Further investigations lead to the establishment of yeast culture preparation's ability to adsorb

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 aflatoxin 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 aflatoxin up to 200ppb level

Mycotoxin binding ability of Maannanoligosaccharides has been demonstrated in various *in vitro* trials (Devegowda et al., 1998) and *in vivo* trials (Raju & Devegowda, 2000; Swamy et

ability either to destroy or transform mycotoxins successfully (Phillips et al., 1988).

(OA), T-2 toxin and zearalenone (ZEA) (Linderfelser & Ceigler, 1970).

**8.6.1 Bacterial degradation**

vomitoxin of 2.10ppm.

1984).

**8.6.2 Protozoan degradation**

**8.6.3 Fungal degradation**

**8.6.4 Degradation by yeast**

degradation (Cooney, 1980).

(Devegowda et al., 1998).

al., 2004).

radicals, reacts with aflatoxin (Dvorak, 1989).

aflatoxins in poultry feeds (Devegowda et al., 1995).

The addition of aflatoxin in layer and breeder hens could lead to aflatoxicosis which can adversely affect the performance and reproductive efficacy of the birds. 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 up to millions of dollars. In commercial Layers, the most prominent manifestations of experimental aflatoxicosis are reduced egg production and egg weight, increased liver fat and alterations in some serum biochemical parameters.In case of breeder hens, many of parameters like changes in body weight, feed consumption, feed conversion ratio, egg production, egg weight, shell thickness, Haugh unit score, yolk color index, fertility, hatchability and embryonic mortality and some of biochemical and immunological parameters like serum levels of GGT and ALT, visceral organ weight and organ lesions and serum antibody titers for ND and IBD could be altered due to aflatoxicosis. This chapter briefly reviews the impact of aflatoxicosis in commercial layer and breeder hens.

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type breeders and their amelioration. Ph.D. thesis submitted to Tamil Nadu

induced liver tumors by antioxidants with increased activity of microsomal

Goncales, E. & Albuquerque, R. (2004). Effects of prolonged administration of aflatoxin B1 and fumonisin B1 in laying Japanese quail. *Poult. Sci.,* 83: 1953-1958. Oguz, H. (2011). A review from experimental trials on detoxification of aflatoxin in poultry

Albuquerque, S.R. & Moro, M.E.G. (2002). Effect of low levels of dietary aflatoxin B1

tissues and eggs of layer chickens fed on diets with various concentrations of

**12**

*India*

**Aflatoxins and Aflatoxicosis in**

**Human and Animals**

D. Dhanasekaran1, S. Shanmugapriya1, N. Thajuddin1 and A. Panneerselvam2

*Bharathidasan University, Tiruchirappalli,*

*1Department of Microbiology, School of Life Sciences,*

*2P.G. & Research Department of Botany & Microbiology,*

*A.V.V.M. Sri Pushpam College, (Autonomous), Poondi, Thanjavur District, Tamil Nadu*

Moldy feed toxicosis was recognized as a serious livestock problem in the 1950's but it was only in 1960 during the investigations in the United Kingdom of moldy feed toxicosis which was called Turkey "x" disease, that *A. flavus* and *A. parasiticus* were identified as the organisms responsible for the elaboration of the toxin in the feed. The earliest symptoms of the disease are lithargy and muscular weakness followed by death. The term aflatoxin now refers to group of bisfuranocoumarin metabolites isolated from strains of *A. flavus* group of fungi. The toxic material derived from the fungus *A. flavus* was given the name "aflatoxin" in

Aflatoxins fluoresce strongly in ultra violet light. The major members are designated as B1, B2, G1 and G2. B1 and B2 fluoresces blue, while Gl and G2 fluoresces green. In some animal species in dairy cattle, aflatoxin B1 and B2 are partially metabolized to the hydroxylated

Aflatoxin P1 is a urinary metabolite of Bl in monkeys. All aflatoxins absorb UV light in the range of 362-363nm, a characteristic used in preliminary identification. The growth of toxigenic molds and elaboration of the toxin occurs if moisture conditions are ideal

Although initially aflatoxin was detected in the peanut meal it is now known that a variety of cereals, and other plant products are susceptible to fungal invasion and mycotoxin production. The occurrence of aflatoxins in agricultural commodities depends on such factors as region, season and the conditions under which a particular crop is grown,

Because of the wide spread nature of fungi producing aflatoxins in food materials, international agencies have now permitted the presence of 20 ppb of aflatoxin in food materials as the maximum permissible level. In 1993 aflatoxin by the World Health Organization (WHO) for cancer research institutions designated as a Class 1 carcinogen, is a highly poisonous toxic substances. Aflatoxin is harmful to human and animal liver tissue

**1. Introduction**

1962 (Sargeant *et al.,* 1963).

following harvest and storage.

harvested or stored.

derivates namely M1 and M2, respectively.

### **Aflatoxins and Aflatoxicosis in Human and Animals**

D. Dhanasekaran1, S. Shanmugapriya1,

N. Thajuddin1 and A. Panneerselvam2

*1Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli,*

*2P.G. & Research Department of Botany & Microbiology,*

*A.V.V.M. Sri Pushpam College, (Autonomous), Poondi, Thanjavur District, Tamil Nadu India*

#### **1. Introduction**

220 Aflatoxins – Biochemistry and Molecular Biology

Swamy, H.V.L.N., Smith, T.K., Karrow, N.A. & Boermans, H.J. (2004). Effects of feeding

Thapa, N.K. (2008). Pathological effects of aflatoxicosis in layer chicken with special

Tiwari, R.P., Virdi, J.S., Gupta, L.K., Saini, S.S. & Vadehra, D.V. (1989). Development of

Trenholm, H.L., Charmley, L.L. & Prelusky, D.B. (1996). Mycotoxin binding agents: An

Verma, J., Johri, T.S., Swan, B.K. & Ameena, S. (2004). Effect of graded levels of aflatoxin and

Wang, R., Miao, R., Chong, N., Sawatwiroj, K., Dawson, S., Fu & Feng, D., 2003. Feed and

Washburn, K.W., Wyatt, R.D., Potts, P.L. & Lanze, G.M., 1985. Effects and mechanism of

Weiss, W.P. (2002). Amntioxidants nutrients and milk quality. In: *Roche symposium, pacific Northwest nutrition conference,* Vancouver, British Columbia, Canada. Wolzak, A., Pearson, A.M., Coleman, T.H., Pestka, J.J. & Gray, J.I. (1985). Aflatoxin

Xue, C.Y., Wang, G.H., Chen, F., Zhang, X.B. & Cao, Y.C. (2010). Immunopathological

Yegani, M., Smith, T. K., Leeson, S. & Boermans, H.J. (2006). Effects of Feeding Grains

Zaghini, A., Martelli, G., Ronchada, P. & Rizzi, L. (2005). Mannanoligosaccharides and

aflatoxin variation in shell strength. *Poult. Sci.,* 64: 1302-1305.

Metabolism of Broiler Breeders*. Poult. Sci.,* 85: 1541-1549.

immunological parameters of broiler chickens. *Poult. Sci.,* 83: 533-543. Sypecka, Z., Kelly, M. & Brereton, P. (2004). Deoxynivalenol and zearalenone residues in

*Chem.*, 52: 5463–5471.

1478.

2176-2182.

1061.

1166.

*Sci.,* 45: 512-518.

*17th Annual Asia Pacific Lecture Tour.*

Veterinary and Animal Sciences University.

blends of grains naturally contaminated with Fusarium mycotoxins on growth and

eggs of laying hens fed with a naturally contaminated diet: Effects on egg production and estimation of transmission rates from feed to eggs*. J. Agric. Food*

emphasis on reproductive pathology. M.V.Sc. Thesis submitted to Tamil Nadu

chicks exposed to aflatoxin B1 during embryogenesis. *Indian J. Anim. Sci.,* 59: 1473-

update on what we know. In: *Biotechnology in the Feed Industry*. Eds: Lyons, T.P. and Jacques, K.A., In: *Proc. Alltech's 17th Annual Symp., KY, USA,* pp. 327-349. Trucksess, M.W., Stoloff, L. & Young, K. (1983). Aflaoxicol and aflatoxins B1 and M1 in eggs

and tissues of laying hens consuming aflatoxin contaminated feed. *Poult. Sci.,* 62:

their combination on the performance and immune response of broilers. *Br. Poult.*

feed ingredient mycotoxin contamination in China: A survey. In: *Proc. of Alltech's*

deposition and clearance in the eggs of laying hens. *Food Chem. Toxixo.,* 32: 1057-

Effects of Ochratoxin A and T-2 Toxin Combination on Broilers. *Poult. Sci.,* 89: 1162-

Naturally Contaminated with *Fusarium* Mycotoxins on Performance and

aflatoxin B1 and M1 residues in fed of laying hens. Effect on egg quality, aflatoxin B1 and M1 residue in eggs and aflatoxin B1 levels in liver. *Poult. Sci.,* 84: 825-832.

Moldy feed toxicosis was recognized as a serious livestock problem in the 1950's but it was only in 1960 during the investigations in the United Kingdom of moldy feed toxicosis which was called Turkey "x" disease, that *A. flavus* and *A. parasiticus* were identified as the organisms responsible for the elaboration of the toxin in the feed. The earliest symptoms of the disease are lithargy and muscular weakness followed by death. The term aflatoxin now refers to group of bisfuranocoumarin metabolites isolated from strains of *A. flavus* group of fungi. The toxic material derived from the fungus *A. flavus* was given the name "aflatoxin" in 1962 (Sargeant *et al.,* 1963).

Aflatoxins fluoresce strongly in ultra violet light. The major members are designated as B1, B2, G1 and G2. B1 and B2 fluoresces blue, while Gl and G2 fluoresces green. In some animal species in dairy cattle, aflatoxin B1 and B2 are partially metabolized to the hydroxylated derivates namely M1 and M2, respectively.

Aflatoxin P1 is a urinary metabolite of Bl in monkeys. All aflatoxins absorb UV light in the range of 362-363nm, a characteristic used in preliminary identification. The growth of toxigenic molds and elaboration of the toxin occurs if moisture conditions are ideal following harvest and storage.

Although initially aflatoxin was detected in the peanut meal it is now known that a variety of cereals, and other plant products are susceptible to fungal invasion and mycotoxin production. The occurrence of aflatoxins in agricultural commodities depends on such factors as region, season and the conditions under which a particular crop is grown, harvested or stored.

Because of the wide spread nature of fungi producing aflatoxins in food materials, international agencies have now permitted the presence of 20 ppb of aflatoxin in food materials as the maximum permissible level. In 1993 aflatoxin by the World Health Organization (WHO) for cancer research institutions designated as a Class 1 carcinogen, is a highly poisonous toxic substances. Aflatoxin is harmful to human and animal liver tissue

Aflatoxins and Aflatoxicosis in Human and Animals 223

The formation of aflatoxins is influenced by physical, chemical and biological factors. The physical factors include temperature and moisture. The chemical factors include the composition of the air and the nature of the substrate. Biological factors are those associated

Specific nutrients, such as minerals (especially zinc), vitamins, fatty acids, amino acids and energy source (preferably in the form of starch) are required for aflatoxins formation (Wyatt, 1991). Large yield of aflatoxins are associated with high carbohydrate concentrations, such as wheat rice and to a lesser extent in oilseeds such as cottonseed, soyabean and peanuts

The limiting temperatures for the production of aflatoxins by *A. flavus* and *A. parasiticus* are reported as 12 to 41°C, with optimum production occurring between 25 and 32°C (Lillehoj, 1983). Synthesis of aflatoxins in feeds are increased at temperatures above 27°C (80 F), humidity levels greater than 62% and moisture levels in the feed above 14% (Royes and

Crops grown under warm and moist weather in tropical or subtropical countries are especially more prone to aflatoxin contamination than those in temperate zones. Groundnuts and groundnut meal are by far the two agricultural commodities that seem to have the highest risk of aflatoxin contamination. Although these commodities are important as substrates, fungal growth and aflatoxin contamination are the consequence of interactions among the fungus, the host and the environment. The appropriate combination of these factors determines the infestation and colonization of the substrate and the type and amount

Water stress, high-temperature stress and insect damage of the host plant are major determining factors in mould infestation and toxin production. Similarly, specific crop growth stages, poor fertility, high crop densities and weed competition have been associated with increased mould growth and toxin production. The moisture content of the substrate and temperature are the main factors regulating the fungal growth and toxin formation. A moisture content of 18% for starchy cereal grains and 9-10% for oil-rich nuts and seeds has been established for maximum production of the toxin (WHO, 1979). On the other hand, the minimum, optimum and maximum temperatures for aflatoxin production have been reported to be 120 - 270C and 400-420C respectively. Frequent contamination of corn and other commodities with high levels of aflatoxins has been a serious problem all over the world resulting in significant economic losses to farmers and a health hazard to farm

In 1963, Asao *et al*.; Van Dorp *et al*. and Van der Zijden characterized the chemical and physical nature of the aflatoxins B1, B2, G1 and G2. Chemically, aflatoxins are difurocoumarolactones (difurocoumarin derivatives). Their structure consists of a bifuran ring fused to a coumarin nucleus with a pentenone ring (in B and M aflatoxins) or a sixmembered lactone ring in G aflatoxins (Fig. 2). The four compounds are separated by the color of their fluorescence under long wave (Devero, 1999) ultraviolet illumination (B=blue,

**2.2 Favorable conditions for aflatoxin biosynthesis production**

with the host species (Hesseltine, 1983).

(Diener and Davis, 1968).

**2.3 Causes of aflatoxin**

of aflatoxin produced.

animals and humans as well.

**2.4 Structure of aflatoxin**

Yanong, 2002).

has damaging effects, serious, can lead to liver cancer or even death. In the natural food contaminated with aflatoxin B1 is most common, is also its most toxic and carcinogenic.

#### **2. Occurence of aflatoxin in food and feed**

Aflatoxin found in soil, plants and animals, all kinds of nuts, especially peanuts and walnuts. In soybean, rice (Fouzia Begum & Samajpati, 2000), corn, pasta, condiments, milk, dairy products, edible oil products are also often found aflatoxin. Aflatoxins often occur in crops in the field prior to harvest. Post harvest contamination can occur if crop drying is delayed and during storage of the crop if water is allowed to exceed critical values for the mould growth. Insect or rodent infestations facilitate mould invasion of some stored commodities.

Aflatoxins are found occasionally in milk, cheese, peanuts, cottonseed (Fig. 1), nuts, almonds, figs, spices, and a variety of other foods and feeds. Milk, eggs, and meat products are sometimes contaminated because of the animal consumption of aflatoxin contaminated feed. Cottonseed, Brazil nuts, copra, various tree nuts and pistachio nuts are the other commodities quite susceptible to the invasion of aflatoxin producing fungi.

Fig. 1. Contamination of cotton seeds

#### **2.1 Types of aflatoxins**

Although 17 aflatoxins have been isolated (WHO, 1979), only 4 of them are well known and studied extensively from toxicological point of view. Being intensely fluorescent in ultraviolet light the four are designated by letters B1, B2, G1 and G2 representing their blue and green fluorescence in UV light. Two other familiar aflatoxins are M1 and M2. Because of their presence in milk of animals previously exposed to B1 and B2. Of all the above-named aflatoxins, aflatoxin B I (AFB1) is the most acutely toxic to various species.

Toxigenic *A. flavus* isolates generally produce only aflatoxins B1 and B2, whereas *A. parasiticus* isolates generally produce aflatoxins B1, B2, G1 and G2 (Davis and Diener, 1983). Other metabolites B2a, aflatoxicol, aflatoxicol H1 and aflatoxins P1 and Q1 have been identified (FDA, 1979). Aflatoxin M1 is a metabolite of aflatoxin B1 in humans and animals. Aflatoxin M2 is a metabolite of aflatoxin B1 in milk of cattle fed on contaminated foods. Although aflatoxins B1, B2 and G1 are common in the same food sample, AFB1 predominates (60-80% of the total aflatoxin content). Generally AFB2, AFG1 and AFG2 do not occur in the absence of AFB1. In most cases AFG1 is found in higher concentrations than AFB2 and AFG2 (Weidenborner, 2001).

has damaging effects, serious, can lead to liver cancer or even death. In the natural food contaminated with aflatoxin B1 is most common, is also its most toxic and carcinogenic.

Aflatoxin found in soil, plants and animals, all kinds of nuts, especially peanuts and walnuts. In soybean, rice (Fouzia Begum & Samajpati, 2000), corn, pasta, condiments, milk, dairy products, edible oil products are also often found aflatoxin. Aflatoxins often occur in crops in the field prior to harvest. Post harvest contamination can occur if crop drying is delayed and during storage of the crop if water is allowed to exceed critical values for the mould growth.

Aflatoxins are found occasionally in milk, cheese, peanuts, cottonseed (Fig. 1), nuts, almonds, figs, spices, and a variety of other foods and feeds. Milk, eggs, and meat products are sometimes contaminated because of the animal consumption of aflatoxin contaminated feed. Cottonseed, Brazil nuts, copra, various tree nuts and pistachio nuts are the other

Although 17 aflatoxins have been isolated (WHO, 1979), only 4 of them are well known and studied extensively from toxicological point of view. Being intensely fluorescent in ultraviolet light the four are designated by letters B1, B2, G1 and G2 representing their blue and green fluorescence in UV light. Two other familiar aflatoxins are M1 and M2. Because of their presence in milk of animals previously exposed to B1 and B2. Of all the above-named

Toxigenic *A. flavus* isolates generally produce only aflatoxins B1 and B2, whereas *A. parasiticus* isolates generally produce aflatoxins B1, B2, G1 and G2 (Davis and Diener, 1983). Other metabolites B2a, aflatoxicol, aflatoxicol H1 and aflatoxins P1 and Q1 have been identified (FDA, 1979). Aflatoxin M1 is a metabolite of aflatoxin B1 in humans and animals. Aflatoxin M2 is a metabolite of aflatoxin B1 in milk of cattle fed on contaminated foods. Although aflatoxins B1, B2 and G1 are common in the same food sample, AFB1 predominates (60-80% of the total aflatoxin content). Generally AFB2, AFG1 and AFG2 do not occur in the absence of AFB1. In most cases AFG1 is found in higher concentrations than

aflatoxins, aflatoxin B I (AFB1) is the most acutely toxic to various species.

Insect or rodent infestations facilitate mould invasion of some stored commodities.

commodities quite susceptible to the invasion of aflatoxin producing fungi.

**2. Occurence of aflatoxin in food and feed**

Fig. 1. Contamination of cotton seeds

AFB2 and AFG2 (Weidenborner, 2001).

**2.1 Types of aflatoxins**

#### **2.2 Favorable conditions for aflatoxin biosynthesis production**

The formation of aflatoxins is influenced by physical, chemical and biological factors. The physical factors include temperature and moisture. The chemical factors include the composition of the air and the nature of the substrate. Biological factors are those associated with the host species (Hesseltine, 1983).

Specific nutrients, such as minerals (especially zinc), vitamins, fatty acids, amino acids and energy source (preferably in the form of starch) are required for aflatoxins formation (Wyatt, 1991). Large yield of aflatoxins are associated with high carbohydrate concentrations, such as wheat rice and to a lesser extent in oilseeds such as cottonseed, soyabean and peanuts (Diener and Davis, 1968).

The limiting temperatures for the production of aflatoxins by *A. flavus* and *A. parasiticus* are reported as 12 to 41°C, with optimum production occurring between 25 and 32°C (Lillehoj, 1983). Synthesis of aflatoxins in feeds are increased at temperatures above 27°C (80 F), humidity levels greater than 62% and moisture levels in the feed above 14% (Royes and Yanong, 2002).

#### **2.3 Causes of aflatoxin**

Crops grown under warm and moist weather in tropical or subtropical countries are especially more prone to aflatoxin contamination than those in temperate zones. Groundnuts and groundnut meal are by far the two agricultural commodities that seem to have the highest risk of aflatoxin contamination. Although these commodities are important as substrates, fungal growth and aflatoxin contamination are the consequence of interactions among the fungus, the host and the environment. The appropriate combination of these factors determines the infestation and colonization of the substrate and the type and amount of aflatoxin produced.

Water stress, high-temperature stress and insect damage of the host plant are major determining factors in mould infestation and toxin production. Similarly, specific crop growth stages, poor fertility, high crop densities and weed competition have been associated with increased mould growth and toxin production. The moisture content of the substrate and temperature are the main factors regulating the fungal growth and toxin formation. A moisture content of 18% for starchy cereal grains and 9-10% for oil-rich nuts and seeds has been established for maximum production of the toxin (WHO, 1979). On the other hand, the minimum, optimum and maximum temperatures for aflatoxin production have been reported to be 120 - 270C and 400-420C respectively. Frequent contamination of corn and other commodities with high levels of aflatoxins has been a serious problem all over the world resulting in significant economic losses to farmers and a health hazard to farm animals and humans as well.

#### **2.4 Structure of aflatoxin**

In 1963, Asao *et al*.; Van Dorp *et al*. and Van der Zijden characterized the chemical and physical nature of the aflatoxins B1, B2, G1 and G2. Chemically, aflatoxins are difurocoumarolactones (difurocoumarin derivatives). Their structure consists of a bifuran ring fused to a coumarin nucleus with a pentenone ring (in B and M aflatoxins) or a sixmembered lactone ring in G aflatoxins (Fig. 2). The four compounds are separated by the color of their fluorescence under long wave (Devero, 1999) ultraviolet illumination (B=blue,

Aflatoxins and Aflatoxicosis in Human and Animals 225

The spectral characteristics of the aflatoxins have been determined by several investigators and are summarized in Table 2. The ultraviolet absorption spectra are very similar, each showing maxima at 223, 265 and 363 mμ. The molar extinction coefficients at the latter two peaks, however, demonstrate that B1 and G2 absorb more intensely than G1 and B2 at these two wavelengths. Because of the close similarities in structural configuration, the infrared absorption spectra of the four compounds are also very similar, as illustrated. The fluorescence emission maximum for B1 and B2 has been reported to be 425 mμ and that for G1 and G2 is 450 mµ. The intensity of light emission, however, varies greatly among the four compounds, a property of significance in the estimation of concentrations of the

) Infrared absorption (cm-1) max

The chemical reactivity and behavior of the aflatoxins has received relatively little systematic study beyond work associated with structure elucidation. However, it has been shown (Asao *et al.,* 1963) that catalytic hydrogenation of aflatoxin B1 to completion results in the uptake of 3 moles of hydrogen with the production of the tetrahydrodeoxy derivative. Interruption of the hydrogenation procedure after the uptake of 1 mole of hydrogen results

Aflatoxin B1 has also been reported to react additively with a hydroxyl group under the catalytic influence of a strong acid (Andrellos *et al*., 1964). Treatment with formic acidthionyl chloride, acetic acid thionyl chloride or trifluroacetic acid results in addition products of greatly altered chromatographic properties, but relatively unchanged fluorescence characteristics. Ozonolysis results in fragmentation of aflatoxin B1 and the products of this reaction include levulinic, succinic, malonic and glutaric acids (Van Drop *et al.,* 1963). The presence of the lactone ring makes the compound labile to alkaline hydrolysis, and partial recyclization after acidification of the hydrolysis product has been reported (De

Although few systematic studies have been carried out on the stability of the aflatoxins, the general experience would seem to indicate that some degradation takes place under several conditions. The compounds appear partially to decompose, for example, upon standing in methanolic solution, and this process is greatly accelerated in the presence of light or heat. Substantial degradation also occurs on chromatograms exposed to air and ultraviolet or visible light. These processes may give rise to some of the nonaflatoxin fluorescent compounds typically seen in chromatograms of culture extracts. The nature of the decomposition products is still unknown, and the chemical reactions involved in their

(mµ) 265

*CHCl*<sup>3</sup> *v*

1562

Fluorescence emission

compounds by fluorescence techniques.

Table 2. Spectral properties of aflatoxin

**2.4.2 Chemical properties**

Iongh *et al*., 1962).

Ultraviolet absorption (

mµ 363 mµ

in the production of aflatoxin B2 in quantitative yield.

formation remain to be established (Wogan *et al*., 1966).

Aflatoxin

B1 B2 G1 G2

G= green). Two other aflatoxins M1 and M2 were isolated from urine and milk and identified as mammalian metabolites of AFB1 and AFB2 (Patterson *et al*., 1978).

Fig. 2. Structure of aflatoxin

#### **2.4.1 Physical properties**

These four compounds were originally isolated by groups of investigators in England (Nesbitt *et al.,*1962, Sargeant *et al.,* 1961). The molecular formula of aflatoxin B1 was established as C17H1206 and of aflatoxin G1 as C17H1207; aflatoxins B2 and G2 were found to be the dihydro derivatives of the parent compounds, C17H1406 and C17H1407 (Hartley *et al*., 1963). Some physical properties of the compounds are summarized in following Table 1.

\*Decomposes

Table 1. Physical properties of aflatoxin

G= green). Two other aflatoxins M1 and M2 were isolated from urine and milk and identified

These four compounds were originally isolated by groups of investigators in England (Nesbitt *et al.,*1962, Sargeant *et al.,* 1961). The molecular formula of aflatoxin B1 was established as C17H1206 and of aflatoxin G1 as C17H1207; aflatoxins B2 and G2 were found to be the dihydro derivatives of the parent compounds, C17H1406 and C17H1407 (Hartley *et al*., 1963). Some physical properties of the compounds are summarized in following Table 1.

> Molecular Weight

Melting Point

268-269\* 286-289\* 244-246\* 237-240\*

C [α]D23

as mammalian metabolites of AFB1 and AFB2 (Patterson *et al*., 1978).

Fig. 2. Structure of aflatoxin

**2.4.1 Physical properties**

B1 B2 G1 G2

\*Decomposes

Aflatoxin Molecular

Table 1. Physical properties of aflatoxin

Formula

C16H12O6 C17H14O6 C17H12O7 C17H14O7 The spectral characteristics of the aflatoxins have been determined by several investigators and are summarized in Table 2. The ultraviolet absorption spectra are very similar, each showing maxima at 223, 265 and 363 mμ. The molar extinction coefficients at the latter two peaks, however, demonstrate that B1 and G2 absorb more intensely than G1 and B2 at these two wavelengths. Because of the close similarities in structural configuration, the infrared absorption spectra of the four compounds are also very similar, as illustrated. The fluorescence emission maximum for B1 and B2 has been reported to be 425 mμ and that for G1 and G2 is 450 mµ. The intensity of light emission, however, varies greatly among the four compounds, a property of significance in the estimation of concentrations of the compounds by fluorescence techniques.

Table 2. Spectral properties of aflatoxin

#### **2.4.2 Chemical properties**

The chemical reactivity and behavior of the aflatoxins has received relatively little systematic study beyond work associated with structure elucidation. However, it has been shown (Asao *et al.,* 1963) that catalytic hydrogenation of aflatoxin B1 to completion results in the uptake of 3 moles of hydrogen with the production of the tetrahydrodeoxy derivative. Interruption of the hydrogenation procedure after the uptake of 1 mole of hydrogen results in the production of aflatoxin B2 in quantitative yield.

Aflatoxin B1 has also been reported to react additively with a hydroxyl group under the catalytic influence of a strong acid (Andrellos *et al*., 1964). Treatment with formic acidthionyl chloride, acetic acid thionyl chloride or trifluroacetic acid results in addition products of greatly altered chromatographic properties, but relatively unchanged fluorescence characteristics. Ozonolysis results in fragmentation of aflatoxin B1 and the products of this reaction include levulinic, succinic, malonic and glutaric acids (Van Drop *et al.,* 1963). The presence of the lactone ring makes the compound labile to alkaline hydrolysis, and partial recyclization after acidification of the hydrolysis product has been reported (De Iongh *et al*., 1962).

Although few systematic studies have been carried out on the stability of the aflatoxins, the general experience would seem to indicate that some degradation takes place under several conditions. The compounds appear partially to decompose, for example, upon standing in methanolic solution, and this process is greatly accelerated in the presence of light or heat. Substantial degradation also occurs on chromatograms exposed to air and ultraviolet or visible light. These processes may give rise to some of the nonaflatoxin fluorescent compounds typically seen in chromatograms of culture extracts. The nature of the decomposition products is still unknown, and the chemical reactions involved in their formation remain to be established (Wogan *et al*., 1966).

Aflatoxins and Aflatoxicosis in Human and Animals 227

(Schabort and Steyn, 1969). Since aflatoxin inhibits protein synthesis, it is conceivable why aflatoxin reduces resistance of poultry to infection with *Pasteurella multocida, salmonella sp.,* Marek disease virus, Coccidia and *Candida albicans* (Smith *et al*., 1969; Hamilton and Harris, 1971). Another effect of aflatoxin is that it causes anticoagulation of blood. This is probably because AFB1 inhibits synthesis of factors II and VII involved in prothrombin synthesis and

Aflatoxin molecules in the double-furan ring structure, the structure is an important toxicity. Studies show that aflatoxin cytotoxic effects of toxins, is an interference RNA and DNA synthesis of information, thereby interfere with cell protein synthesis, resulting in systemic damage to animals (Nibbelink, 1988). Huangguang Qi *et al.,* (1993) research indicates that aflatoxin B1 to form with the tRNA adduct, aflatoxin-tRNA adduct can inhibit tRNA binding activity of some amino acids on protein synthesis in the essential amino acids such as lysine, leucine, arginine and glycine and tRNA binding, have different inhibitory effect,

Aflatoxin B1 is excreted in urine and feces, and also in milk of lactating animals either unchanged or as various metabolites (Nabney *et al*., 1967; Allcroft *et al*., 1968). Only one milk metabolite, namely AFM1, appears to be the major metabolite of AFB1 that has shown appreciable oral toxicity (Holzapfel *et al*., 1966). Its toxicity is considered to be nearly as potent as AFB1. Even so this metabolite may be detoxified by conjugation with taurocholic and glucuronic acids prior to excretion in the bile or brine (DeIongh *et al*., 1964; Bassir and Osiyemi, 1967). In this respect, two other metabolites of AFB1, namely, AFP1 and AFQ1 are similar in that they also undergo this type of detoxication (Dalezios *et al*., 1971; Buchi *et al*., 1973; Masri *et al*., 1974). Both of these metabolites are several-fold less toxic than AFB1. For example, toxicity tests showed that AFP1 causes some mortality in newborn mice at 150 mg/kg as compared to the LD50 of 9.5 mg/kg for AFB1 under

*CHCl*<sup>3</sup> *v* interfering with the translation level of protein biosynthesis, affect cell

clotting mechanism (Bababunmi and Bassir, 1969).

Fig. 3. Metabolism of aflatoxin in liver

thereby max

metabolism.

comparable conditions.

#### **2.5 Biological effects of the aflatoxins**

The discovery of this group of compounds as contaminants of animal feeds and the potential public health hazards involved, have stimulated considerable research effort concerned with their effects in various biological assay systems. The toxic properties of the aflatoxins manifest themselves differently depending on the test system, dose and duration of exposure. Thus, they have been shown to be lethal to animals and animal cells in culture when administered acutely in sufficiently large doses and to cause histological changes in animals when smaller doses were administered subacutely. Chronic exposure for extended periods has resulted in tumor induction in several animal species (Wogan *et al*., 1966).

#### **2.5.1 Aflatoxin and animal diseases**

Aflatoxin poisoning (Aflatoxicosis) mainly on animal liver injury, the injured individual species of animals, age, gender and nutritional status vary. The aflatoxin can cause liver dysfunction, reduced milk production and egg production and to reduced immunity of animals. Susceptible to the infection of harmful microorganisms. In addition, long-term consumption of food containing low concentrations of aflatoxin in feed can also result in embryo toxicity. Usually young animals are more sensitive to aflatoxin. The clinical manifestations of aflatoxin are digestive disorders, reduced fertility, reduced feed efficiency and anemia. Aflatoxins not only the decline in milk production, but also the transformation of the milk containing aflatoxin M1 and M2. According to the U.S. agricultural economy scientists to statistics, the consumption of aflatoxin contaminated feed, make at least 10% of the U.S. livestock industry suffered economic losses.

#### **2.5.2 Metabolism and mechanisms of action of aflatoxin B1**

The absorption from the gastrointestinal tract should be complete since very small doses, even in the presence of food, can cause toxicity. After the absorption, highest concentration of the toxin is found in the liver (Mintzlaff *et al*., 1974). Once in liver, aflatoxin B1 is metabolized by microsomal enzymes to different metabolites through hydroxylation, hydration, demethylation and epoxidation (Fig. 3). Thus hydroxylation of AFB1 at C4 or C22 produces, AFM1 and AFQ1, respectively. Hydration of the C2 – C3 double bond results in the formation of AFB2a which is rapidly formed in certain avian species (Patterson and Roberts, 1970). AFP1 results from o-demethylation while the AFB1 – epoxide is formed by epoxidation at the 2,3 double bond. Aflatoxicol is the only metabolite of AFB1 produced by a soluble cytoplasmic reductase enzyme system.

The liver is the target organ for toxic effects of aflatoxin B1. As a result, metabolism of proteins, carbohydrates and lipids in liver is seriously impaired by AFB1. The toxin inhibits RNA polymerase and subsequent protein synthesis at a faster rate in ducks than in rats probably because of faster liver metabolism of AFB1 in ducks than in rats (Smith, 1965). In day-old chicks, AFB1 reduces the activity of liver UDP glucose-glycogen transglucosylase resulting in depletion of hepatic glycogen stores (Shankaran *et al*., 1970). On the other hand, there is lipid accumulation in the liver of chickens and ducklings exposed to aflatoxin (Carnaghan *et al*., 1966; Shank and Wogan, 1966). With regard to its toxic effects on liver microsomal enzymes, AFB1 is known to decrease microsomal glucose-6-phosphatase activity (Shankaran *et al*., 1970) whereas stimulation of microsomal enzyme activity by inducers seems to be unaffected by AFB1 (Kato *et al*., 1970). In fact, pretreatment with the toxin actually stimulates its own metabolism in the rat when this is assayed in vitro

The discovery of this group of compounds as contaminants of animal feeds and the potential public health hazards involved, have stimulated considerable research effort concerned with their effects in various biological assay systems. The toxic properties of the aflatoxins manifest themselves differently depending on the test system, dose and duration of exposure. Thus, they have been shown to be lethal to animals and animal cells in culture when administered acutely in sufficiently large doses and to cause histological changes in animals when smaller doses were administered subacutely. Chronic exposure for extended periods has resulted in tumor induction in several animal species (Wogan *et*

Aflatoxin poisoning (Aflatoxicosis) mainly on animal liver injury, the injured individual species of animals, age, gender and nutritional status vary. The aflatoxin can cause liver dysfunction, reduced milk production and egg production and to reduced immunity of animals. Susceptible to the infection of harmful microorganisms. In addition, long-term consumption of food containing low concentrations of aflatoxin in feed can also result in embryo toxicity. Usually young animals are more sensitive to aflatoxin. The clinical manifestations of aflatoxin are digestive disorders, reduced fertility, reduced feed efficiency and anemia. Aflatoxins not only the decline in milk production, but also the transformation of the milk containing aflatoxin M1 and M2. According to the U.S. agricultural economy scientists to statistics, the consumption of aflatoxin contaminated feed, make at least 10% of

The absorption from the gastrointestinal tract should be complete since very small doses, even in the presence of food, can cause toxicity. After the absorption, highest concentration of the toxin is found in the liver (Mintzlaff *et al*., 1974). Once in liver, aflatoxin B1 is metabolized by microsomal enzymes to different metabolites through hydroxylation, hydration, demethylation and epoxidation (Fig. 3). Thus hydroxylation of AFB1 at C4 or C22 produces, AFM1 and AFQ1, respectively. Hydration of the C2 – C3 double bond results in the formation of AFB2a which is rapidly formed in certain avian species (Patterson and Roberts, 1970). AFP1 results from o-demethylation while the AFB1 – epoxide is formed by epoxidation at the 2,3 double bond. Aflatoxicol is the only metabolite of AFB1 produced by

The liver is the target organ for toxic effects of aflatoxin B1. As a result, metabolism of proteins, carbohydrates and lipids in liver is seriously impaired by AFB1. The toxin inhibits RNA polymerase and subsequent protein synthesis at a faster rate in ducks than in rats probably because of faster liver metabolism of AFB1 in ducks than in rats (Smith, 1965). In day-old chicks, AFB1 reduces the activity of liver UDP glucose-glycogen transglucosylase resulting in depletion of hepatic glycogen stores (Shankaran *et al*., 1970). On the other hand, there is lipid accumulation in the liver of chickens and ducklings exposed to aflatoxin (Carnaghan *et al*., 1966; Shank and Wogan, 1966). With regard to its toxic effects on liver microsomal enzymes, AFB1 is known to decrease microsomal glucose-6-phosphatase activity (Shankaran *et al*., 1970) whereas stimulation of microsomal enzyme activity by inducers seems to be unaffected by AFB1 (Kato *et al*., 1970). In fact, pretreatment with the toxin actually stimulates its own metabolism in the rat when this is assayed in vitro

**2.5 Biological effects of the aflatoxins**

**2.5.1 Aflatoxin and animal diseases**

the U.S. livestock industry suffered economic losses.

a soluble cytoplasmic reductase enzyme system.

**2.5.2 Metabolism and mechanisms of action of aflatoxin B1**

*al*., 1966).

(Schabort and Steyn, 1969). Since aflatoxin inhibits protein synthesis, it is conceivable why aflatoxin reduces resistance of poultry to infection with *Pasteurella multocida, salmonella sp.,* Marek disease virus, Coccidia and *Candida albicans* (Smith *et al*., 1969; Hamilton and Harris, 1971). Another effect of aflatoxin is that it causes anticoagulation of blood. This is probably because AFB1 inhibits synthesis of factors II and VII involved in prothrombin synthesis and clotting mechanism (Bababunmi and Bassir, 1969).

Fig. 3. Metabolism of aflatoxin in liver

Aflatoxin molecules in the double-furan ring structure, the structure is an important toxicity. Studies show that aflatoxin cytotoxic effects of toxins, is an interference RNA and DNA synthesis of information, thereby interfere with cell protein synthesis, resulting in systemic damage to animals (Nibbelink, 1988). Huangguang Qi *et al.,* (1993) research indicates that aflatoxin B1 to form with the tRNA adduct, aflatoxin-tRNA adduct can inhibit tRNA binding activity of some amino acids on protein synthesis in the essential amino acids such as lysine, leucine, arginine and glycine and tRNA binding, have different inhibitory effect, thereby max *CHCl*<sup>3</sup> *v* interfering with the translation level of protein biosynthesis, affect cell metabolism.

Aflatoxin B1 is excreted in urine and feces, and also in milk of lactating animals either unchanged or as various metabolites (Nabney *et al*., 1967; Allcroft *et al*., 1968). Only one milk metabolite, namely AFM1, appears to be the major metabolite of AFB1 that has shown appreciable oral toxicity (Holzapfel *et al*., 1966). Its toxicity is considered to be nearly as potent as AFB1. Even so this metabolite may be detoxified by conjugation with taurocholic and glucuronic acids prior to excretion in the bile or brine (DeIongh *et al*., 1964; Bassir and Osiyemi, 1967). In this respect, two other metabolites of AFB1, namely, AFP1 and AFQ1 are similar in that they also undergo this type of detoxication (Dalezios *et al*., 1971; Buchi *et al*., 1973; Masri *et al*., 1974). Both of these metabolites are several-fold less toxic than AFB1. For example, toxicity tests showed that AFP1 causes some mortality in newborn mice at 150 mg/kg as compared to the LD50 of 9.5 mg/kg for AFB1 under comparable conditions.

Aflatoxins and Aflatoxicosis in Human and Animals 229

The disease was characterized by high fever, high colored urine, vomiting, and edema of feet, Jaundice, rapidly developing ascitis, portal hypertension and a high mortality rate. The disease was confirmed to the very poor, who were forced by economic circumstances to consume badly molded corn containing aflatoxins between 6.25 -15.6 ppm, an average daily intake per person of 2-6 mg of aflatoxins (Krishnamachari *et al*., 1975a and 1975b; Keeler and

Long exposure to aflatoxins in the diet increases risk with a synergistic effect from increased alcohol consumption. Af1atoxin B1 has also been implicated as a cause of human hepatic cell carcinoma (HCC). Aflatoxin B1 also chemically binds to DNA and caused structural DNA

Ingestion of aflatoxin, viral diseases, and hereditary factors has been suggested as possible aetiological agents of childhood cirrhosis. There are evidences to indicate that children exposed to aflatoxin breast milk and dietary items such as unrefined groundnut oil, may develop cirrhosis. Malnourished children are also prone to childhood cirrhosis on consumption of contaminated food. Several investigators have suggested aflatoxin as an aetiological agent of Reye's syndrome in children in Thailand, New Zealand etc. Though there is no conclusive evidence as yet. Epidemiological studies have shown the involvement of aflatoxins in Kwashiorkor mainly in malnourished children. The diagnostic features of Kwashiorkor are edema, damage to liver etc. These out breaks of aflatoxicosis in man have been attributed to ingestion of contaminated food such as maize, groundnut etc. Hence it is

Aflatoxin can cause oncogenesis, chronic toxicity or peracute signs depending on the species, age of animal, dose and duration of aflatoxin exposure (Smith, 2002). All animal species are susceptible to aflatoxicosis, but outbreak occurs mostly in pigs, sheep and cattle (Radostits *et al*., 2000). Beef and dairy cattle are more susceptible to aflatoxicosis than sheep or horses. Young animals of all species are more susceptible than mature animals to the effects of aflatoxin. Pregnant and growing animals are less susceptible than young animals, but more susceptible than mature animals (Cassel *et al*., 1988). Nursing animals may be affected by exposure to aflatoxin metabolites secreted in the milk (Jones *et al.,* 1994). For most species, oral LD50 values of aflatoin B1 vary from 0.03 to 18 mg/kg

There are differences in species with respect to their susceptibility to aflatoxins, but in

Acute toxicity is less likely than chronic toxicity. Studies have shown that ducklings are the species most susceptible to acute poisoning by aflatoxins. The LD50 of a day old duckling is

general, most animals, including humans, are affected in the same manner.

alterations with the result of genomic mutation (Groopman *et al*, 1985).

very important to reduce the dietary intake of aflatoxins.

**2.8 Effect on human health**

**2.8.1 Acute toxicity**

**2.8.2 Chronic toxicity**

**2.9 Aflatoxicosis in animals**

**2.10 Effects on animal health**

**2.10.1 Acute toxicity**

0.3mg/kg bodyweight.

body weight.

Tu, 1983).

#### **2.6 Aflatoxin and human health**

Human health hazards by aflatoxin were mainly due to people eating aflatoxin contaminated food. For the prevention of this pollution is very difficult, the reason is due to fungi in the food or food materials in the presence of a very common. The state health department has been heavily polluted enterprises to use against the grain for food production and supervision of enterprises to develop the implementation of the relevant standards. But with lower concentrations of aflatoxin food and food cannot be controlled. In developing countries, consumption of aflatoxin contaminated food and incidence of cancer was positively correlated. Asian and African research institutions, disease research showed that aflatoxin in food and liver cells cancer (Liver Cell Cancer, LCC) showed positive correlation. For a long time with low concentrations of aflatoxin consumption of food was the leading cause liver cancer, stomach cancer, colon cancer and other diseases. In 1988, International Agency for Research on Cancer (IARC) classified the aflatoxin B1 as a human carcinogen.

The median lethal dose of aflatoxin Bl 0.36 mg / kg body weight is a special range of highly toxic poison (aflatoxin animal half of the lethal dose is found in the strongest carcinogens). Its carcinogenicity is 900 times more than dimethylnitrosamine induced liver cancer in the large capacity 75 times higher than the 3,4-benzopyrene, a large 4000-fold. It is mainly to induce liver cancer in animals, can also induce cancer, renal cancer, colorectal cancer and breast, ovary, small intestine and other sites of cancer.

#### **2.7 Aflatoxicosis**

The disease caused by the consumption of substances or foods contaminated with aflatoxin is called aflatoxicosis.

#### **2.7.1 Aflatoxicosis in humans**

#### **2.7.2 Human exposure conditions**

Two pathways of the dietary exposure have been identified:

Aflatoxin M1 is believed to be associated with casein (protein) fraction of milk. Cream and butter contain lower concentration of M1 than the milk from which these products are made, while, cheese contains higher concentration of M1 about 3-5 times the M1 in the original milk (Kiemeier and Buchner, 1977; Stoloff, 1980; Brackett and Marth, 1982).

The expression of aflatoxin related diseases in humans may be influenced by factors such as age, sex, nutritional status, and concurrent exposure to other causative agents such as viral hepatitis (HBV) or parasite infestation.

#### **2.8 Effect on human health**

#### **2.8.1 Acute toxicity**

228 Aflatoxins – Biochemistry and Molecular Biology

Human health hazards by aflatoxin were mainly due to people eating aflatoxin contaminated food. For the prevention of this pollution is very difficult, the reason is due to fungi in the food or food materials in the presence of a very common. The state health department has been heavily polluted enterprises to use against the grain for food production and supervision of enterprises to develop the implementation of the relevant standards. But with lower concentrations of aflatoxin food and food cannot be controlled. In developing countries, consumption of aflatoxin contaminated food and incidence of cancer was positively correlated. Asian and African research institutions, disease research showed that aflatoxin in food and liver cells cancer (Liver Cell Cancer, LCC) showed positive correlation. For a long time with low concentrations of aflatoxin consumption of food was the leading cause liver cancer, stomach cancer, colon cancer and other diseases. In 1988, International Agency for Research on

The median lethal dose of aflatoxin Bl 0.36 mg / kg body weight is a special range of highly toxic poison (aflatoxin animal half of the lethal dose is found in the strongest carcinogens). Its carcinogenicity is 900 times more than dimethylnitrosamine induced liver cancer in the large capacity 75 times higher than the 3,4-benzopyrene, a large 4000-fold. It is mainly to induce liver cancer in animals, can also induce cancer, renal cancer, colorectal cancer and

The disease caused by the consumption of substances or foods contaminated with aflatoxin

a. Direct ingestion of aflatoxins (mainly B1) in contaminated foods of plant origin such as

b. Ingestion of aflatoxins carried over from feed into milk and milk products including cheese and powdered milk, where they appear mainly as aflatoxin M1. In addition to the carryover into milk, residues of aflatoxins may be present in the tissues of animals that consume contaminated feed (WHO, 1979). Aflatoxin residues have been found in animal tissues, eggs and poultry following the experimental ingestion of aflatoxin-contaminated feed (Rodricks and stoloff, 1977). Contamination of milk, egg and meat can result from animal consumption of mycotoxin contaminated feed. Aflatoxins, ochratoxin and some trichothecences have been given considerable attention, because they are either carcinogenic or economic concern in animal health

Aflatoxin M1 is believed to be associated with casein (protein) fraction of milk. Cream and butter contain lower concentration of M1 than the milk from which these products are made, while, cheese contains higher concentration of M1 about 3-5 times the M1 in the original

The expression of aflatoxin related diseases in humans may be influenced by factors such as age, sex, nutritional status, and concurrent exposure to other causative agents such as viral

milk (Kiemeier and Buchner, 1977; Stoloff, 1980; Brackett and Marth, 1982).

Cancer (IARC) classified the aflatoxin B1 as a human carcinogen.

breast, ovary, small intestine and other sites of cancer.

Two pathways of the dietary exposure have been identified:

**2.7 Aflatoxicosis**

is called aflatoxicosis.

(CAST, 1989).

**2.7.1 Aflatoxicosis in humans 2.7.2 Human exposure conditions**

maize nuts and their products.

hepatitis (HBV) or parasite infestation.

**2.6 Aflatoxin and human health**

The disease was characterized by high fever, high colored urine, vomiting, and edema of feet, Jaundice, rapidly developing ascitis, portal hypertension and a high mortality rate. The disease was confirmed to the very poor, who were forced by economic circumstances to consume badly molded corn containing aflatoxins between 6.25 -15.6 ppm, an average daily intake per person of 2-6 mg of aflatoxins (Krishnamachari *et al*., 1975a and 1975b; Keeler and Tu, 1983).

#### **2.8.2 Chronic toxicity**

Long exposure to aflatoxins in the diet increases risk with a synergistic effect from increased alcohol consumption. Af1atoxin B1 has also been implicated as a cause of human hepatic cell carcinoma (HCC). Aflatoxin B1 also chemically binds to DNA and caused structural DNA alterations with the result of genomic mutation (Groopman *et al*, 1985).

Ingestion of aflatoxin, viral diseases, and hereditary factors has been suggested as possible aetiological agents of childhood cirrhosis. There are evidences to indicate that children exposed to aflatoxin breast milk and dietary items such as unrefined groundnut oil, may develop cirrhosis. Malnourished children are also prone to childhood cirrhosis on consumption of contaminated food. Several investigators have suggested aflatoxin as an aetiological agent of Reye's syndrome in children in Thailand, New Zealand etc. Though there is no conclusive evidence as yet. Epidemiological studies have shown the involvement of aflatoxins in Kwashiorkor mainly in malnourished children. The diagnostic features of Kwashiorkor are edema, damage to liver etc. These out breaks of aflatoxicosis in man have been attributed to ingestion of contaminated food such as maize, groundnut etc. Hence it is very important to reduce the dietary intake of aflatoxins.

#### **2.9 Aflatoxicosis in animals**

Aflatoxin can cause oncogenesis, chronic toxicity or peracute signs depending on the species, age of animal, dose and duration of aflatoxin exposure (Smith, 2002). All animal species are susceptible to aflatoxicosis, but outbreak occurs mostly in pigs, sheep and cattle (Radostits *et al*., 2000). Beef and dairy cattle are more susceptible to aflatoxicosis than sheep or horses. Young animals of all species are more susceptible than mature animals to the effects of aflatoxin. Pregnant and growing animals are less susceptible than young animals, but more susceptible than mature animals (Cassel *et al*., 1988). Nursing animals may be affected by exposure to aflatoxin metabolites secreted in the milk (Jones *et al.,* 1994). For most species, oral LD50 values of aflatoin B1 vary from 0.03 to 18 mg/kg body weight.

#### **2.10 Effects on animal health**

There are differences in species with respect to their susceptibility to aflatoxins, but in general, most animals, including humans, are affected in the same manner.

#### **2.10.1 Acute toxicity**

Acute toxicity is less likely than chronic toxicity. Studies have shown that ducklings are the species most susceptible to acute poisoning by aflatoxins. The LD50 of a day old duckling is 0.3mg/kg bodyweight.

Aflatoxins and Aflatoxicosis in Human and Animals 231

due to the formation of 8,9-epoxide, which binds to DNA and alters gene expression. There is a correlation with the presence of aflatoxins and increased liver cancer in individuals that are hepatitis B carriers. Animals of different species vary in their susceptibility to acute aflatoxin

In fact duckling liver metabolized aflatoxin very rapidly in vitro (Patterson and Allcroft, 1970), although the species is sufficiently susceptible for day old birds to be used widely in a sensitive bioassay for the toxin (Patterson, 1973). Studies indicated that rabbit, duckling and guinea-pig constitute a "fast metabolizing group" being apparently capable of handling an LD50 dose in under 12 minutes. Chick, mouse, pig and sheep fall into an intermediate group, metabolizing an LD50 dose in a few hours. So far, the rat is the only example of a "slow metabolizing group" in which LD50 dose would probably disappear from the liver over a

Factors that influence aflatoxin toxicity residue levels in animal species include: species and breeds of animals and poultry, levels and duration of exposure, nutrition and health of

**Toxin Animal Age/Size LD50(mg/kg)**

AFB2 1.69(84.8µg/50gm

AFG2 2.5(172.5µg/duckling) AFM1 0.8(16.6µg/duckling)

> Rabbit 0.3-0.5 Cat 0.55 Pig 6.0-7.0 kg 0.62 Turkey 0.5-1.0 Dog Puppies 0.5-1.0 Cattle Young calves 0.5-1.0

> > Young foals

Rat male 100 gm 17.9

21 days 5.5

Horse 2.0 Sheep 2.0 Monkey 2.2 Chickens 6.5-16.5 Mouse 9.0 Hamster 10.2

0.37

duckling)

1.4-2.0

7.4

Duckling Day old

AFG1 0.79

Table 4. A comparison of single oral LD50 values for AFB1 in various species. (Agag, 2004)

Aflatoxin ingested in the feed by cattle is physically bound to ruminal contents, and as little as 2-5% reach the intestine. Levels of AFB1 in excess of 100 μg/kg of feed are considered to be poisonous for cattle (Radostits *et al*., 2000). The effects of aflatoxin fed to cattle depend on the level of aflatoxin in the ration, the length of feeding period and the age of animal (Jones

poisoning with LD50 values ranging from 0.3 to 17.9 mg/kg (Table 4).

animals, age, sex and diseases, drugs and other mycotoxins (FDA, 1979).

Guinea pig

Rat, male, female

period of days (Patterson, 1973).

AFB1

AFB1

*et al*., 1994).

**2.10.4 Aflatoxicosis in ruminants**

Table 3. Comparative LD50 or lethal values for Aflatoxin B1 (Edds, 1973 &WHO, 1979).

The principal target organ for aflatoxins is the liver. After the invasion of aflatoxins into the liver, lipids infiltrate hepatocytes and leads to necrosis or liver cell death. The main reason for this is that aflatoxin metabolites react negatively with different cell proteins, which leads to inhibition of carbohydrate and lipid metabolism and protein synthesis. In correlation with the decrease in liver function, there is a derangement of the blood clotting mechanism, icterus (jaundice), and a decrease in essential serum proteins synthesized by the liver. Other general signs of aflatoxicosis are edema of the lower extremities, abdominal pain, and vomiting.

#### **2.10.2 Chronic toxicity**

Animals which consume sub-lethal quantities of aflatoxin for several days or weeks develop a sub acute toxicity syndrome which commonly includes moderate to severe liver damage. Even with low levels of aflatoxins in the diet, there will be a decrease in growth rate, lowered milk or egg production and immunosupression. There is some observed carcinogenicity, mainly related to aflatoxin B1. Liver damage is apparent due to the yellow color that is characteristic of jaundice, and the gall bladder will become swollen. Immunosuppression is due to the reactivity of aflatoxins with T-cells, decrease in Vitamin K activities and a decrease in phagocytic activity in macrophages.

#### **2.10.3 Cellular effects**

Aflatoxins are inhibitors of nucleic acid synthesis because they have a high affinity for nucleic acids and polynucleotides. They attach to guanine residues and form nucleic acid adducts. Aflatoxins also have been shown to decrease protein synthesis, lipid metabolism, and mitochondrial respiration. They also cause an accumulation of lipids in the liver, causing a fatty liver. This is due to impaired transport of lipids out of the liver after they are synthesized. This leads to high fecal fat content. Carcinogenisis has been observed in rats, ducks, mice, trout, and subhuman primates and it is rarely seen in poultry or ruminants. Trout are the most susceptible. In fact, 1ppb of aflatoxin B1 will cause liver cancer in trout. Carcinogenisis occurs

(mg/Kg)

0.025 0.3 0.5 2.0 6.3 5.0 7.2 17.9 0.3 0.6 0.6 1.4 10.2 9.0 2.0

Species Oral LD50/Lethal dose

Table 3. Comparative LD50 or lethal values for Aflatoxin B1 (Edds, 1973 &WHO, 1979).

The principal target organ for aflatoxins is the liver. After the invasion of aflatoxins into the liver, lipids infiltrate hepatocytes and leads to necrosis or liver cell death. The main reason for this is that aflatoxin metabolites react negatively with different cell proteins, which leads to inhibition of carbohydrate and lipid metabolism and protein synthesis. In correlation with the decrease in liver function, there is a derangement of the blood clotting mechanism, icterus (jaundice), and a decrease in essential serum proteins synthesized by the liver. Other general signs of aflatoxicosis are edema of the lower extremities, abdominal pain, and

Animals which consume sub-lethal quantities of aflatoxin for several days or weeks develop a sub acute toxicity syndrome which commonly includes moderate to severe liver damage. Even with low levels of aflatoxins in the diet, there will be a decrease in growth rate, lowered milk or egg production and immunosupression. There is some observed carcinogenicity, mainly related to aflatoxin B1. Liver damage is apparent due to the yellow color that is characteristic of jaundice, and the gall bladder will become swollen. Immunosuppression is due to the reactivity of aflatoxins with T-cells, decrease in Vitamin K

Aflatoxins are inhibitors of nucleic acid synthesis because they have a high affinity for nucleic acids and polynucleotides. They attach to guanine residues and form nucleic acid adducts. Aflatoxins also have been shown to decrease protein synthesis, lipid metabolism, and mitochondrial respiration. They also cause an accumulation of lipids in the liver, causing a fatty liver. This is due to impaired transport of lipids out of the liver after they are synthesized. This leads to high fecal fat content. Carcinogenisis has been observed in rats, ducks, mice, trout, and subhuman primates and it is rarely seen in poultry or ruminants. Trout are the most susceptible. In fact, 1ppb of aflatoxin B1 will cause liver cancer in trout. Carcinogenisis occurs

activities and a decrease in phagocytic activity in macrophages.

Chick embryo Duckling Turkey poultry

Sheep Rat(male) Rat(female) Rabbit Cat Pig

Guinea pig Hamster Mouse Baboon

vomiting.

**2.10.2 Chronic toxicity**

**2.10.3 Cellular effects**

Chicken, New Hampshire Chicken, Rhode Island

due to the formation of 8,9-epoxide, which binds to DNA and alters gene expression. There is a correlation with the presence of aflatoxins and increased liver cancer in individuals that are hepatitis B carriers. Animals of different species vary in their susceptibility to acute aflatoxin poisoning with LD50 values ranging from 0.3 to 17.9 mg/kg (Table 4).

In fact duckling liver metabolized aflatoxin very rapidly in vitro (Patterson and Allcroft, 1970), although the species is sufficiently susceptible for day old birds to be used widely in a sensitive bioassay for the toxin (Patterson, 1973). Studies indicated that rabbit, duckling and guinea-pig constitute a "fast metabolizing group" being apparently capable of handling an LD50 dose in under 12 minutes. Chick, mouse, pig and sheep fall into an intermediate group, metabolizing an LD50 dose in a few hours. So far, the rat is the only example of a "slow metabolizing group" in which LD50 dose would probably disappear from the liver over a period of days (Patterson, 1973).

Factors that influence aflatoxin toxicity residue levels in animal species include: species and breeds of animals and poultry, levels and duration of exposure, nutrition and health of animals, age, sex and diseases, drugs and other mycotoxins (FDA, 1979).

Table 4. A comparison of single oral LD50 values for AFB1 in various species. (Agag, 2004)

#### **2.10.4 Aflatoxicosis in ruminants**

Aflatoxin ingested in the feed by cattle is physically bound to ruminal contents, and as little as 2-5% reach the intestine. Levels of AFB1 in excess of 100 μg/kg of feed are considered to be poisonous for cattle (Radostits *et al*., 2000). The effects of aflatoxin fed to cattle depend on the level of aflatoxin in the ration, the length of feeding period and the age of animal (Jones *et al*., 1994).

Aflatoxins and Aflatoxicosis in Human and Animals 233

Decreased performance (i.e. rate of gain, milk production) is one of the most sensitive indicators of aflatoxicosis (Richard *et al*., 1983). The ultimate cause of this effect is probably multifactorial, involving not only nutritional interactions, but also the compounding influences of anorexia, deranged hepatic protein and lipid metabolism and disturbances in hormonal metabolism (Raisbeck *et al*., 1991). Aflatoxins have shown to affect rumen motility (Cook *et al*., 1986) and rumen function by decreasing cellulose digestion, volatile fatty acid

Anorexia, depression and icterus were observed in sheep and goats exposed to aflatoxin. The goats also developed a nasal discharge and dark brown urine was noted in the sheep

Anorexia and diarrhea occurred in sheep given aflatoxin at a rate of 0.23mg/kg. These signs were accompanied by excessive salivation, tachypnea and pyrexia at dosages of 0.59 mg/kg or more. Heavy mucous diarrhea and dysentery were observed in sheep dosed at a rate of 1.28 to 2.0mg/kg. Sheep that died within 24 hours of dosing had marked centrilobular necrosis of the liver. Sheep that survived until the 7th day after dosing had periportal congestion of the liver, widely dilated sinusoids and necrosis of liver cells (Armbrecht *et al*.,

Acutely intoxicated sheep with 4mg/kg showed anorexia, diarrhea, excessive salivation,

The existing information on aflatoxicosis in the horse is inconclusive, although a total dietary concentration of 500–1000 µg/kg has been shown to induce clinical changes and liver damage, depending on the duration of exposure (Meerdink, 2002). The target organ in horses, as in all affected animals, is the liver, where the toxin induces centrilobular necrosis (Stoloff and Trucksess, 1979). Horses suffering from aflatoxicosis exhibit non-specific clinical signs, such as inappetence, depression, fever, tremor, ataxia and cough (Larsson *et al*., 2003). Necropsy findings include yellow–brown liver with centrilobular necrosis, icterus, haemorrhage, tracheal exudates and brown urine (Angsubhakorn *et al*., 1981; Cysewski *et*

Clinical signs of toxicity were observed in adult male Shetland ponies given a daily oral dose of 0.075 (over 36 or 39 days), 0.15 (over 25 or 32 days) and 0.3 mg/kg (over 12 or 16 days) of partially purified aflatoxin (47% AFB1 activity) (Cysewski *et al*., 1982). Daily clinical observations revealed the appearance of depression, inappetence and weakness 5– 7 days before the onset of severe illness and death. The onset of signs was generally dose related and appeared first in the ponies given 0.3mg/kg. Prothrombin time, sulfabromophthalein clearance time, total plasma bilirubin and the icteric index increased markedly before death. An increase in the plasma activity of aspartate amino transferase (AST) was observed. The AST activity grew significantly and generally remained at elevated levels in all animals.  Microscopic lesions, including centrilobular fatty change, hepatic cell necrosis and periportal fibrosis, were observed in

rumen atony, scour, rectal prolapse, fever and death (Wylie and Morehouse, 1978).

production and proteolysis (Fehr and Delag, 1970; Bodine and Mertins, 1983).

(Hatch *et al*., 1971; Samarajeewa, *et al*., 1975; Abdelsalam *et al*., 1989).

**2.10.7 Sheep and goats**

**2.10.8 AFB1 toxicity in equine species**

*al*., 1982; Bortell *et al*., 1983; Vesonder *et al*., 1991).

all ponies (Cysewski *et al*., 1982).

1970).

#### **2.10.5 Calves**

The LD50 dosage of AFB1 in calves has been estimated to be 0.5-1.5 mg/kg. Affected calves had anorexia, depression, jaundice, photosensitization of unpigmented skin, submandibular edema, severe keratocojunctivitis and diarrhea with dysentery. Collapse and death followed. Postmortem findings showed hemorrhages in subcutaneous tissues, skeletal muscles, lymph nodes, pericardium, beneath the epicardium and serosa of the alimentary tract. The liver was pale and carcass jaundiced. Histopathological examination of the liver revealed that hepatocytes were markedly enlarged, especially in the periportal areas and occasional hepatocyte nuclei were up to 5 times the diameter of their companions. Hepatocyte cytoplasm was finely vacuolated, many of these vacuoles containing fat. Serum enzymes of hepatic origin and bilirubin were elevated.

In calves who have consumed contaminated rations for several weeks, the onset of clinical signs is rapid. The most consistent features are blindness, circling and falling down, with twitching of the ears and grinding of the teeth. Severe tenesmus and erosion of the rectum are seen in most cases, and death of some cases (Humphreys, 1988).

#### **2.10.6 Dairy and beef cattle**

The signs most commonly reported with acute toxicosis in cattle include anorexia, depression, dramatic drop in milk production, weight loss, lethargy, ascitis, icterus, tenesmus, abdominal pain (animals may stretch or kick at their abdomen), bloody diarrhea, abortion, hepatoencephalopathy, photosensitization and bleeding (Colvin *et al*., 1984; Cook *et al*., 1986; Ray *et al*., 1986; Eaton and Groopman, 1994; Reagor, 1996). Other signs associated with acute aflatoxicosis include blindness, walking in circles, ear twitching, frothy at the mouth, keratoconjunctivitis and rectal prolapse (Radostits *et al*., 2000).

Hepatic damage is a constant finding in acute aflatoxicosis. Lesions include fatty degeneration, megalocytosis and single-cell necrosis with increasing fibrosis, biliary proliferation and veno occlusive lesions as the disease progresses (Burnside *et al*., 1957; Morehouse, 1981; Colvin *et al*., 1984).

In additions, chronic aflatoxicosis may impair reproductive efficiency including abnormal estrous cycle (too short and too long) and abortions, induce immunosuppression and increase susceptibility to disease (Cassel *et al*., 1988). The immunotoxic effect of AFB1 was expressed via the cell-mediated immune system (Raisbeck *et al*., 1991).