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Aflatoxins- Hazard to Livestock and Poultry

Production: A Review

Article · January 2007

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 J. Immunol. Immunopathol. 2007. 9(1 & 2): 1-15
Society for Immunology and Immunopathology, Izatnagar

AFLATOXINS- HAZARD TO LIVESTOCK AND POULTRY PRODUCTION:

A REVIEW
K Dhama, RS Chauhan1, Mahesh Mahendran, KP Singh1, AG Telang1, Lokesh Singhal1 and Simmi
Tomar2

Division of Pathology; 1CADRAD, Indian Veterinary Research Institute, Izatnagar-243122 (UP); 2Division of Animal Sciences, Central Agricultural
Research Institute(CARI), Port Blair, A&N Islands, INDIA. E.mail: rschauhan_123@rediffmail.com

ABSTRACT
Dhama K, Chauhan RS, Mahendran M, Singh KP, Telang AG, Singhal L and Tomar S. (2007). Aflatoxins- Hazard to Livestock
and Poultry Production: A Review. J. Immunol. Immunopathol. 9(1 & 2): 1-15.
Aflatoxins are known to be the most dangerous fungal toxins that cause detrimental effects on animal and avian
health. They are hepatotoxic, mutagenic and carcinogenic compounds produced mainly by Aspergillus species, which are
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more prevalent in tropical regions. Among the toxins, aflatoxin B1 is considered as the most potent hepatotoxic agent. Acute
and chronic aflatoxicoses in animals and birds occur due to consumption of aflatoxin contaminated feed, leading to serious
pathological and immune insufficiencies. Humoral and cellular immune functions are impaired, and the primary and
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secondary immune responses are also affected. Aflatoxicosis in animals result in decreased feed utilization and reduced
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productivity. In avian species, immunosuppression, reduced growth and low productivity are commonly seen. For detection
of aflatoxins, chromatographic techniques like TLC and HPLC are commonly used apart from immunochemical detection
and affinity based analysis. Recently, detection of toxins and toxigenic fungi are performed using biosensors and polymerase
chain reaction, respectively. One of the effective measures to be adopted to protect the livestock from aflatoxin exposure is to
establish proper surveillance system and also formulate regulatory levels as these may enable to reduce the incidence of
aflatoxicoses. As a prevention strategy, the most commonly followed method is the detoxification by toxin binding agents or
chemical inactivation. This review is an attempt to highlight the pathological and immunological impairments that the
aflatoxins render to livestock and poultry and to make familiarize the various strategies to detect, prevent and control the
malady.

Keywords: Aflatoxins, aspergillus, carcinogenesis, detoxification, immunosuppression, mycotoxins, poultry, ruminants.

INTRODUCTION
The term ‘mycotoxin’ was coined in 1962 in the aftermath of death of about 100,000 turkey poults in London, due
to “Turkey X disease” in 1960, linked to a peanut meal contaminated with aflatoxins. Now, about 300 to 400 toxic
fungal metabolites have been identified as mycotoxins, of which some are capable of inflicting deleterious effects to
animal and avian health (Singhal and Kaur, 2005). There are three major genera of fungi that produce mycotoxins:
Aspergilius, Fusarium and Penicillium (Bennett and Klich, 2003). Among these, the Aspergillus species are of much
concern as they are responsible for the production of aflatoxins, the most potent fungal toxin causing aflatoxicoses
that have detrimental effects on living beings (Gremmels, 1999; Barret, 2000). Aflatoxicoses tend to be a major
problem in the tropics where high humidity and temperature creates optimal conditions for mould growth (Jelinek et al.,
1989). The financial impact of mycotoxins on intensive livestock production can be of immense magnitude in these
regions. Giving contaminated feeds to animals and birds can lead to reduced growth rates, illness, and death. The
produced products contain toxic residues and the biotransformation products may be harmful to human health
(Yiannikouris and Jouany, 2002; Bintvihok et al., 2002).
One of the most effective measures that has to be adopted to protect the livestock health is to set up proper
surveillance and formulating regulatory levels for aflatoxins (Bennett and Klich, 2003; Jand et al., 2005). Apart from
this, it is of paramount importance to develop rapid and more sensitive advanced techniques to detect the presence of
aflatoxins in feed ingredients (Whittaker, 2003; Zheng et al., 2006). The presence of mycotoxins is often unavoidable,
but stringent measures to prevent and control them and monitor the feed stuffs by set regulations may enable to

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 reduce the incidence of aflatoxicoses. For this, a profound knowledge of aflatoxins, their mode of action, harmful
effects on animal and avian health, prevention and control measures are needed. This review is an attempt to focus
aflatoxicoses associated with animals and birds, with emphasis on its harmful immunonogical and pathological effects
and also to make familiarize the various strategies to minimize the looming threat posed by aflatoxins.

AFLATOXINS
Currently, there are about 20 known aflatoxins but most of these are metabolites that are formed endogenously
in animals by major toxins of A. flavus and A. parasiticus viz. aflatoxin B1, B2, G1, G2, M1 and M2 (‘B’ and ‘G’
denotes blue fluorescence and green fluorescence, and ‘M’ is due to its presence in milk). Aflatoxin is hepatotoxic,
mutagenic and carcinogenic fungal toxin which is capable of producing diseases in farm animals as well as poultry
and is known to be produced by specific moulds in feed commodities (Bennet and Klich, 2003). The ingestion of
aflatoxin contaminated materials is deleterious to animals and birds as they may lead to serious health problems
involving liver in addition to causing immunosuppression and acting as an inciting factor for carcinogenesis (Cary and
Ehrlich, 2006). The toxic effects of aflatoxins are largely due to their different chemical structures and acute effects
require high amounts to be consumed by livestock or domesticated birds. The symptoms depend on the type of toxin,
the amount and duration of the exposure, age, health status and poorly understood synergistic effects involving
genetics, dietary status, and interactions with other toxic insults (Bennett and Klich, 2003).
As per the FAO reports, about 25% of the world’s agricultural production is contaminated with mycotoxins of
which aflatoxins contribute significantly. Aflatoxin development in stored grains has constantly hampered the availability
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of good quality feed ingredients. Contaminated crops are sometimes diverted into animal and poultry feed causing
considerable health and economic implications. The financial impact of mycotoxins on intensive production systems
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can be analyzed from studies conducted, especially in the poultry industry (Beg et al., 2006). Aflatoxins cause
enormous economic losses to poultry industry by decreasing immunity in flocks and enhancing susceptibility to
various infections, decreased egg and meat production, decreased feed consumption, loss of weight gain and nutritional
interactions (Pimpukdee et al., 2004; Jand et al., 2005). Hence, aflatoxin demands much toxicological and mycological
research over other mycotoxins for finding out solutions to the problem that has really threatened and has caused a
negative impact on the scope of animal product exports of many tropical countries. To counter the harmful effects,
most countries have now adopted strict regulations to limit exposure to aflatoxins (Jand et al., 2005).
Types of aflatoxins and their metabolism: Aflatoxins are family of related bisfurano-coumarin compounds produced
mainly by at least three species of Aspergillus namely A. flavus, A. parasiticus and A. nominus (Bennett and Klich,
2003). These are mainly classified in four groups, viz., B1, B2, G1 and G2 (Yiannikouris and Jouany, 2002). Aflatoxins
B2 and G2 are the dihydro derivatives of the parent compound, while M1 and M2 are the hydroxylated metabolites of
aflatoxins B1 and B2, respectively and are produced when ruminants ingest feed contaminated with these toxins.
Aflatoxin in cattle is metabolized and excreted in milk and designated as ‘M’ or milk toxin (Veldman et al., 1992). Of
all the types, B1 is the most predominant and the most toxic one. Two more aflatoxin metabolites viz. P1 and Q1 have
also been identified to occur in liver or urine of mammals. Aflatoxin B2a, G2a and aspertoxin have also been reported
but their pathogenic potential is not fully understood (Yiannikouris and Jouany, 2002; Bennett and Klich, 2003).
Aflatoxins on ingestion are absorbed in small intestines, and they get accumulated in liver and muscles, liver
being the principal organ affected. Hepatocytes metabolize these toxins and convert into various metabolites (Mclean
and Dutton, 1995). The 2-hydroxylation of aflatoxin B1 (AFB1) is the common biotransformation in all animal species
yielding a comparatively non-toxic metabolite designated as aflatoxin B2a, and G1 on 2-hydroxylation gets transformed
into G2a (Yiannikouris and Jouany, 2002). Cyclopentenone hydroxylation of the AFB1 results in the 22-hydroxylation
product, referred as aflatoxin Q1. The metabolism of aflatoxin through O-demethylation occurs in liver of ruminants. In
liver of birds, aflatoxin B1 and B2 are reduced to aflatoxicol and dihydroaflatoxicol, respectively (Verma, 2005). In liver,
AFB1 irreversibly binds to protein and form adducts such as aflatoxin B1-lysine in albumin (Skipper and Tannenbaum,
1990). Epoxidation is another process of metabolism that occurs in liver of mammals and birds.

CLINICOPATHOLOGICAL EFFECTS OF AFLATOXINS

The general effects of aflatoxicosis in animals include decreased feed utilization and efficiency leading to poor
weight gain, liver and kidney damage, gastrointestinal dysfunction, embryonic death, teratogenicity, carcinogenesis,

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 suppressed immune functions, reduced productivity, anaemia, jaundice and death (Devegowda, 1990; Pier, 1992). In
birds, immunosuppression, reduced growth and production potential are often noticed (Yaroshenko et al., 2003).
Acute liver damage, induction of tumors and immunotoxicities are of much concern (Bennett, 1987; Berry, 1988;
Bennet and Klich, 2003). Recently, studies have been carried out to assess the involvement of proteasomes and the
enzyme functionality implications, which gave evidence to the role of aflatoxins in binding to the proteasome and to
generate oxidative stress to the tissues (Amici et al., 2007). High level of aflatoxin exposure produces alteration in
digestion, absorption and metabolism of nutrients. Chronic or subclinical exposure does not lead to visible signs but
leads to carcinogenesis, as the metabolites aflatoxins can intercalate into DNA (Bennett and Klich, 2003). Over a very
long exposure, teratogenic and mutagenic effects can also be observed in livestock (Edds et al., 1980). Embryotoxic
potential of AFB1 and AFM1 has been evaluated using the frog embryo teratogenesis assay-Xenopus laevis blastulae
revealed that when bio-activated with microsome activation systems, AFB1 exerts embryotoxic effects whereas AFM1
is a non-embryotoxic compound (Vismara et al., 2006).
High doses of aflatoxins may also result in severe hepatocellular necrosis and prolonged low dosages result in
reduced growth rate and hepatomegaly (Etzel, 2002; Bennett and Klich, 2003). It inhibits the DNA-dependent RNA
synthesis in nucleus resulting into inhibition of protein synthesis, which results in reduced production of essential
metabolic enzymes and structural proteins for growth (Pier, 1992; Etzel, 2002; Fung and Clark, 2004).
Carcinogenesis: Aflatoxins can induce mutagenesis by alkylation of nuclear DNA, leading to carcinogenesis and
teratogenesis. The reaction with DNA occurs with guanine in the codon 249 of tumor suppressor gene p53 where the
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G to T transversion occurs (Hussain et al., 2007). The International Agency for Research on Cancer (IARC) defines
aflatoxin as a Class I carcinogen, regulated levels being 20 ppb in grains and 0.5 ppb in milk in the United States and
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4 ppb in foods and agricultural commodities in many European countries (Henry et al., 1999). Aflatoxin B1 is one of the
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most potent hepato-carcinogenic substances known and no animal species is immune to its acute toxic effects. The
active metabolite of AFB1, the 8, 9,-epoxide (previously referred to as 2, 3-epoxide), is a potent carcinogen and named
as “proximal carcinogen”. It is extremely reactive, and induces chromosomal aberrations and cell toxicity. Evidence
suggests that this epoxide can also induce mutations in the ras oncogene in experimental animals (Railey et al.,
1997). Aflatoxin G1 is an active carcinogen but aflatoxins B2 and G2 are very less carcinogenic. Aflatoxin M1 has 2, 3-
vinyl ether double-bond, which exerts the carcinogenic effects.
Immunosuppression: Studies on farm animals and avian models have given evidence of the ability of AFB1 to induce
thymic aplasia, reduce T-lymphocyte function and number, and suppress phagocytic and complement activity (Bondy
and Pestka, 2000; Kataria et al., 2005). Aflatoxin impairs the humoral and cellular immune functions thus reducing the
primary and secondary immune responses (Giambrone et al., 1978; Fernandez et al., 2000). AFB1 is also known to
have immunosuppressive effects on NK cell activity (Methenitou et al., 2001). Impairment of cellular function by
aflatoxins seems to be due to its effects on the production of lymphokines (Han et al., 1999). Cusumano et al. (1996)
have reported that AFB1 had a marked impact on phagocytosis by adversely modulating cytokines produced by the
phagocytes. Dugyala and Sharma (1996) reported suppression of the levels of IL-1, IL-6 and TNF by AFB1 and slight
decrease in both mRNA and protein levels of IL-2 and IFN gamma in lymphocytes. Pre-treatment of monocytes with
AFB1 also resulted in reduced release of IL-1, IL-6 and TNF alpha (Kurtz and Czuprynski, 1992; Rossano et al., 1999).
Rossano et al. (1999) reported that AFB1 completely blocked the transcription of IL-1 alpha, IL-6 and TNF alpha
mRNAs. Phytohemagglutinin-stimulated and AFB1 treated blood cells also indicated that aflatoxins decreased
proinflammatory cytokine mRNA expression (Marin et al., 2002). Ghosh et al. (1990a) reported that the percentage of
active lymphocytes significantly decreased in chicks due to AFB1. Chicken embryos exposed to AFB1 showed a
depressed graft-versus-host response (Kadian et al., 1989). Thymic and bursal involution and impairment of delayed
cutaneous hypersensitivity in birds, and suppression of lymphoblastogenesis in bovines have been reported (Bodine et
al., 1984). In birds, experimental studies have proved that the development of bursa is affected in chicks during
aflatoxin exposure (Sur and Celik, 2003; Verma et al., 2004). Aflatoxins also impair the function of macrophages in
avian species (Mohiuddin et al., 1986). Swine that received AFB1 in their feed had reduced total hemolytic serum
complement activity and the immunosuppressive effects were transferred across the porcine placenta to the fetus
(Silvotti et al., 1997). Significantly, aflatoxin exposure also reduces the antibody response to vaccines. In poultry,
dietary aflatoxin exposure reduced antibody titers to vaccines for Newcastle disease, Marek’s disease and infectious
bursal disease (Batra et al., 1991; Anjum, 1994). Decreased effectiveness of vaccination against swine fever and

3
 hemorrhagic septicemia has also been observed in animals due to aflatoxicoses (Singh et al., 1997; Choudhury et al.,
1998).
Nutritional interference: Chronic aflatoxin exposure has major effects on nutritional status in animals. The efficiency
of feed intake is less in animals that are exposed to aflatoxins (Fung and Clark, 2004; Jand et al., 2005). In poultry and
swine, a drop in feed conversion efficiency is observed and decreased growth rates are a consistent sign of chronic
aflatoxin exposure and the toxin has been a factor modulating the rate of recovery from protein malnutrition (Bennett
and Klich, 2003). It has been established through experiments that aflatoxin modifies vitamin A nutrition in poultry,
halving the serum retinol concentrations (Tyczkowski and Hamilton, 1987). Reddy et al. (1989) found that liver vitamin
A decreased with increasing concentrations of aflatoxins in female broiler chicks. In broilers, the vitamin D concentration
is also affected by aflatoxin in the diet. Aflatoxin have been found to reduce plasma 25-hydroxyvitamin D and 1,25-
dihydroxyvitamin D concentrations after a 5 day treatment (Glahn et al., 1991). Zinc and selenium are essential for
healthy immune systems, also get affected by aflatoxin in the diet (Kalorey et al., 1996; Hegazy and Adachi, 2000). In
swine, maternal exposure to aflatoxins approximately halved the serum zinc content in piglets, resulting in decreased
cellular immunity in piglets due to less activated thymulin (Korenev, 1989; Silvotti et al., 1997).

EFFECTS OF AFLATOXINS IN VARIOUS LIVESTOCK AND AVIAN SPECIES:

Aflatoxins generally affect growing poultry, piglets, pregnant sows and calves. Adult cattle, sheep, and goats are
relatively resistant to the acute form of the disease but are susceptible if toxic diets are fed over long periods. Dietary
levels of aflatoxin (in ppb) generally tolerated are =50 in chicks, =100 in adult chicken, =50 in weaner pigs, =200 in
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finishing pigs, <100 in calves, and <300 in adult cattle (Ramdell and Eaton, 1990). But dietary levels as low as 10-20
ppb may result in measurable aflatoxin M1 and M2 being excreted through milk in cattle (Frobish et al., 1986).
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Individual effects of aflatoxins in various livestock and avian species are enlisted.
Cattle: A disease outbreak of aflatoxicosis led to pasteurellosis was reported in an organized frozen semen bull
station with 15 out of 34 deaths. The animals showed the symptoms of staggering gait dullness, depression anorexia
and death. Grossly, animals showed increased amount (3-4 lit) of straw coloured fluid in abdominal and thoracic
cavities. Intestinal mucosa was thickened, oedematous, severely congested and haemorrhagic. The aflatoxin B1 was
detected in wheat straw (1.75 ppm) and feed concentrate (1.50 ppm) (Anonymous, 2006).
Rabbit: Singh et al. (1993) reported the outbreak of aflatoxicosis in rabbit farm involving 23 (15.8%) deaths. Grossly,
liner was enlarged and dark brown to tan in colour. Kidneys, lungs and small intestine revealed congestion.
Histopathologically, foci of coagulative necrosis along with bile ductular hyperplasia, pericellular and periportal cirrhosis
in liner and rarefactive changes in lymphoid cells were noticed in spleen. Singh and Singh (1998) reported the outbreak
of aflatoxicosis in broiler rabbits causing 22.7% mortality. The symptoms of anorexia, dullness, dyspnoea, rough hair
coat, gradual weight loss, diarrhea and emaciation were noticed. The affected rabbits were in active growing stage
between 3 weeks to 9 months of age. The liners were found enlarged, dark brown to tan in colour with grayish white
foci. Other visceral organs were found congested.
Ruminants: Animals are generally sensitive to aflatoxins but ruminants are considered relatively resistant. In the
order of increasing susceptibility, domestic livestock can be classified as sheep, goat, cattle and pigs (Ramdell and
Eaton, 1990). However, protein deficiency increases the susceptibility of animals to aflatoxicosis (Richardson et al.,
1987a). Early signs of toxicoses include reduction in feed intake, weight loss or decreased rate of weight gain (Bodine
and Mertens, 1990). Other effects in dairy and beef cattle are decreased feed efficiency, immunosuppression, increased
susceptibility to stress, and decreased reproductive performances (Rajendran et al., 1992; Bennet and Klich, 2003).
There is growing evidence for anti-reproductive effects of aflatoxin in ruminants, including decreased fertility in sheep,
and abortion and birth of underweight calves in cattle (Diekman and Green, 1992). Milk production may be decreased
in dairy cows and aflatoxins may pass through milk causing toxicities in calves (Corbett et al., 1988).
In cattle, the manifestations of acute aflatoxicoses are blindness with frequent falling, walking in circles, twitching
of ear, grinding of teeth, diarrhea, rectal prolapse, tenesmus, convulsions and death (Adam, 1988; Bodine and Mertens,
1990; Cockroft, 1995). In cattle, AFB1 reduces milk production and impairs the growth of calves. In pregnant animals,
abortions are common (Diekman and Green, 1992). In calves, there is retarded growth rate and they may succumb

4
 within 48 hrs of acute exposure (Colvin et al., 1984). The level of aflatoxin exceeds 100 ppb is considered toxic for
cattle and signs of chronic exposure are dry nose, harsh coat, inappetence, abdominal pain and diarrhoea (Applibaum
and Marth, 1983; Adam, 1988). The gross lesions include ascites, visceral oedema, fibrosis of liver with dark red
colour and distended gall bladder with thin dark bile (Rajendran et al., 1992). Microscopically, there is proliferation of
bile duct epithelium, obliterating endophlebitis of centrilobular and hepatic veins (characteristic to bovines) and diffuse
fibrosis leading to variation in morphology of hepatocytes. Hepatocytes show granularity with infiltration of lymphocytes
and neutrophils (Rajendran et al., 1992). In case of buffaloes aflatoxicosis is manifested as ataxia, staggering gait,
grinding of teeth, emaciation and recumbency, and lesions include fatty changes in liver, bile duct hyperplasia and
extensive fibrosis (Galhotra et al., 1986). The serum alkaline phosphatase, bilirubin, aspartate amino transferase and
cholesterol levels increases in buffaloes while there is a decrease in serum proteins and clotting time.
Sheep is considered quite resistant and even feeding 1 mg/kg body weight dose of aflatoxin B1 does not produce
any lesions but, nasal carcinoma and chondroma have been reported (Abdelsalam et al., 1989). AFB1 at 4 mg/kg body
weight may cause acute toxicosis, characterized by inappetence, weakness and lethargy, excessive salivation, atony
of rumen, blood and mucous mixed dung. Liver tumors have been seen in sheep on prolonged feeding of aflatoxin for
3-5 years (Lewis et al., 1967). In lambs, loss of weight gain and performance along with alteration in hematological
values have been reported (Fernandez et al., 2000). Lesions in affected sheep include congestion and dilatation of
sinusoids, periportal lymphocytic cell infiltration, hepatic carcinoma, purulent metritis and chondroma and neoplasms
in nasal cavity (Sulaiman et al., 1987). Aflatoxicosis in goats is manifested by decreased feed consumption, loss of
body weight, mucopurulent nasal discharge, dyspnoea, lethargy, icterus, diarrhoea, decreased T-lymphocytes in
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blood and reduced delayed type hypersensitivity (DTH) reaction to phytohaemagglutinin (Clark et al., 1984). There is
increase in total erythrocyte count, packed cell volume, haemoglobin, bilirubin, and serum enzymes (Miller et al.,
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1984). Microscopic lesions visualized were bile duct proliferation, hepatocytic karyomegaly and hepatocellular
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degeneration (Clark et al., 1984).

Swine: Decreased feed efficiency and poor weight gain are the mildest effects in swine. Severe toxicity results in
acute hepatitis, systemic haemorrhages, nephrosis and death (Coppock et al., 1989). Piglets are more sensitive with
stunted growth (Yang et al., 1988). Major clinical signs in swine are depression, muscular weakness, shivering,
inappetence, fever followed by subnormal temperature, bloody diarrhoea and convulsions. Gross lesions include
atrophy of gall bladder, tan-coloured liver, haemorrhages in the intestines, free blood in lumen of intestines, icteric
pericardium, ascites, petechiae on fat, muscles and subcutaneous tissue (Cysewski et al., 1968; Harvey et al., 1990).
An increase in serum ornithine carbamyl transferase and alkaline phosphatase and a decrease in total serum protein,
blood pressure and total leucocyte count were observed (Gill et al., 1986). Aflatoxicosis may cause upto 20% mortality
in piglets, characterized by enterocolitis, diarrhoea, dysentery, abortions, depression, fever, icterus, ascites and
elevation of serum enzyme levels (Blevins et al., 1969). Immune system is suppressed which leads to decreased
resistance to infectious diseases (Silvotti et al., 1997).
Birds: Avian species are variably sensitive to chronic aflatoxicosis (Arafa et al., 1981) and the intake occur via
mycotoxin contaminated feeds (Fraga et al., 2007). Turkey poultrys and ducklings are the most sensitive (Lozano and
Diaz, 2006). Aflatoxins are highly immunosuppressive and increase the susceptibility of birds to many infectious
agents (Dalvi, 1986). The productive performance of birds is also drastically affected (Yaroshenko et al., 2003).
Aflatoxicosis in chickens is manifested as lethargy, loss of appetite, decreased feed consumption and growth rate,
diarrhoea, ophisthotonus, spasms of neck muscles, arched back and death with legs stretched posteriorly (Asuzu
and Shetty, 1986; Borisova et al., 1990). Due to immunosuppressive damage of thymus and bursa of Fabricius, birds
become susceptible to infections and fail to induce protective antibody titres (Kataria et al., 2005; Jand et al., 2005).
In layers, there is drop in egg production and poor hatchability (Jand et al., 2005). Acute aflatoxicosis cause sudden
death with enlarged mottled liver and extensive bile duct proliferation while chronic exposure of aflatoxins may result
in low mortality reduced growth rate, pale firm liver and pale enlarged kidneys (Asuzu and Shetty, 1986). There is
congestion of myocardium, catarrhal inflammation of intestines, distension of pericardial sac and haemorrhages in
pancreas and serous membranes (Espada et al., 1992). The gross pathological lesions include enlarged liver with pale
necrotic lesions and distended gall bladder (Bhalla, 1982; Asuzu and Shetty, 1986; Jand et al., 2005). In chronic
cases, cirrhosis of liver is more marked and birds may have gelatinous transudate subcutaneously. The microscopic
lesions include degenerative and necrotic changes in liver with infiltration of polymorphonuclear and lymphocytic cells

5
 and bile duct hyperplasia (Espada et al., 1992). Accumulation of fat in liver may also be seen in birds died due to
aflatoxicosis (Fukal et al., 1988). In kidneys, there is thickening of capillary basement membrane in glomeruli, ischemia,
swollen tubules and multiple and diffuse haemorrhages in parenchyma (Glahn et al., 1991).
In turkeys, the clinical signs and lesions include ruffled feathers, diarrhoea, ataxia, opisthotonus, convulsions,
swollen feet, keratoconjunctivitis and generalized edema (Fazal et al., 1980). The immune system of poults is highly
compromised during aflatoxicosis (Quist et al., 2000). The liver becomes edematous and orange tinted in acute cases
while in chronic condition liver becomes pale, and shrunken (Quist et al., 2000). Gross lesions in poults are subcutaneous
haemorrhages, enlargement of kidneys, catarrhal enteritis, impaction and distension of gizzard and myocardial
congestion. On microscopic examination of liver, eosinophilic patches of degeneration in hepatocytes with hyperplastic
bile duct may be seen. In kidneys, there is thickening of glomerular basement membrane and hyaline degeneration of
tubular epithelium. Desquamation of epithelial cells in mucosa of duodenum, granular myocardial degeneration in
heart and lymphoid depletion in spleen were also observed (Richard et al., 1973; Giambrone et al., 1985).
The characteristic signs of aflatoxicosis in ducks and ducklings include inappetence, poor growth rate, ataxia,
opisthotonus, convulsions and subcutaneous haemorrhages in legs and feet (Asplin and Carnaghan, 1961; Meissner,
1983). Gross lesions include pale enlarged liver with hyperplastic nodules and pale and swollen kidneys with petechial
haemorrhages. Microscopically, cell swelling, vacuolation, nuclear enlargement, karyorrhexis and karyolysis, proliferation
of bile duct epithelium and mild fibrosis were seen in liver (Meissner, 1983). There are also reports that aflatoxicosis
augment the hepatotoxicity in birds infected with duck hepatitis virus (Uchida et al., 1988).
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DETECTION OF AFLATOXINS
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As a traditional method, bioassays using ducklings, chicken embryos and brine shrimp larvae were used for
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toxicity testing of feed ingredients (Watson and Lindsay, 1982; Celik et al., 2000). But, now the detection largely
depends on chromatographic techniques and immunochemical detection using sophisticated equipments. All methods
of detection have common extraction followed by a clean up before separation by various chromatographic methods
(Schiefer, 1990). Immunoaffinity columns (IAC) are widely used for cleanup and isolation of aflatoxins (Chu, 1992;
Stroka et al., 2000; Castegnaro et al., 2006). Recently, Sobolev (2007) has developed a rapid and inexpensive clean up
procedure using minicolumn packed with florisil. After extraction and clean up, thin layer chromatography (TLC) is
used to identify and quantitate aflatoxins at very low levels (Dutta and Das, 2001; Grosso et al., 2004). Gas
chromatography (GC) is also used to elute toxic compounds and to detect them specifically (Richard et al., 1993;
Sforza et al., 2006). Another widely used method is high performance liquid chromatography (HPLC) but it requires
costly equipments and standardization of the laboratory (Yen and Bidasec, 1993). HPLC using fluorescence detection
has recently become the most accepted method for the determination of aflatoxins (Blesa et al., 2005). Rapid
immunochemical screening methods like enzyme-linked immunosorbent assay (ELISA) and immunoaffinity column
assay (ICA) have been developed and are commercially available (Schiefer, 1990). Now, ELISA has been routinely
used for detection of Aflatoxin B1 in cereals and animal tissues (Dutta and Das, 2001; Gathumbi et al., 2003; Lee et
al., 2004). Monoclonal antibody-based ELISA of AFB1 has been developed and is utilized for specific detection of the
toxin (Fenelon et al., 1999). Chauhan (1997) and Lipigorngoson et al. (2003) have reported the usefulness of Dot
immunobinding assay and competitive ELISA respectively, for detection of aflatoxins. An antibody-colloidal gold probe
conjugate has been developed recently for rapid and specific detection of AFB1 where the detection take less time as
compared to ELISA technique and the lower test limit was found to be around 2.5 ng/ml (Xiulan et al., 2005). The
optical waveguide lightmode spectroscopy (OWLS) has also been used for the detection of aflatoxins in immunoassays
(Adanyi et al., 2007).
Rapid minicolumn method for aflatoxin detection in agricultural products has also been developed which is less
expensive (Arim et al., 1999). el Nagerabi and Elshafie (2001) developed a method for the detection of aflatoxins in
grain, known as the Bright greenish-yellow fluorescence (BGYF) test. In this method, feed samples are inspected
under UV light for a characteristic bright greenish yellow fluorescence in damaged kernels. Flaherty et al. (1995) have
reported the use of a reporter gene to monitor gene expression and presence of A. flavus. Currently, mass spectroscopy
(MS) in combination with liquid chromatography is becoming popular for identification of aflatoxins (Sulyok et al.,
2006; Cavaliere et al., 2007). It has high specificity, good reliability and wide possibilities for automation. Recently,

6
 Saha et al. (2007) have reported the use of catalyzed reporter deposition method of signal amplification called “tyramide
signal amplification”, which can be used in immunoassays to increase the specificity of aflatoxin detection and to
reduce the assay time. Recent and advanced techniques for the detection of toxins and toxigenic fungi include
biosensors and polymerase chain reaction (PCR) (Zheng et al., 2006). Biosensor assay works on the surface plasmon
resonance (SPR) and monoclonal antibody technique based detection (Zheng and Binder, 2005; Logrieco et al.,
2005). Other sensor systems including fibre-optic sensors and chemiluminescence are currently being investigated
(Logrieco et al., 2005). MicroSERS is a new biochip technology that uses surface-enhanced Raman scattering (SERS)
microscopy for label-free transduction and detection of aflatoxins (Grow et al., 2003). Molecular detection of Aspergillus
using PCR has also been reported by many workers (Makimura et al., 1994; Skladny et al., 1999), in which primers for
aflatoxin regulatory (afl R), o-methyltransferase (omt) and oxidoreductase (ord) genes are used. In future, an accurate
level of contamination of aflatoxin in animal food products and other commodities must be obtained by exploiting these
rapid and advanced detection methodologies that would enable us to dwindle down the aflatoxin menace.

PREVENTION AND DETOXIFICATION STRATEGIES

Prevention would be the most effective means of reducing aflatoxin contamination but needs much improvement
in agricultural storage methods, practices in harvesting and handling of crops (Kubena et al., 1987; Chauhan and
Mahipal, 1996). Processes such as dry and wet milling can also result in the distribution of aflatoxin residues into less
utilized fractions in the commodity (Piva et al., 1995). Non-biological materials such as anti-caking agents covalently
bind aflatoxins and diminish aflatoxin uptake, prevent acute aflatoxicosis and reduces aflatoxin residues in animal
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products. Preservatives and anti-fungal agents, such as propionic acid and mould inhibitors like hydroxyquinoline and
gentian violet may be somewhat effective in reducing aflatoxin formation (Rao, 1995). Wilkinson et al. (2003) have
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studied the effect of a mucosal aflatoxin B1 vaccine by coupling it with carrier proteins and found that the immunization
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elicited significant IgG responses. As a common prevention strategy, the method followed is detoxification of the
possibly contaminated feed by toxin binding agents or by chemical inactivation.
Aflatoxins are very heat-stable but are destroyed partially by prolonged heating or autoclaving. The UV irradiation
and gamma-rays (5-10 Mrad) have been reported to stop growth of molds in feeds (Park, 1993). Extraction of aflatoxin
contaminated groundnut meal with 1% calcium chloride yields 75-80% of toxin, while ammoniation can reduce the
concentrations of aflatoxin by greater than 99% (Park, 1993). Aflatoxin B1 can also be degraded by treatment with
sodium hydroxide and sodium bisulfite (Kataria et al., 2005). The genotoxic effects of the aflatoxins can be prevented
by using ellagic acid peracetate (Kumar et al., 2007). Gunterus et al. (2007) have reported that ethylene inhibits
aflatoxin synthesis and can be used to reduce aflatoxin contamination of crops. Apart form these, toxin binding
compounds have the affinity to bind aflatoxins and thus prevent its absorption. Common toxin binders are aluminas,
zeolites, silica, phyllosilicates and chemically modified phyllosilicates (Huwig et al., 2001). Clay and zeolite minerals-
a considerably complex family of aluminosilicates, interact with the aflatoxins through electrostatic forces or by
covalent bond formation (Huwig et al., 2001; Dakovic et al., 2005). Among all, HSCAS (0.5%) has proved to be superior
in protecting from the toxic effects of aflatoxicosis (Kubena et al., 1993).
Biological detoxification is another strategy in which degradation of aflatoxins is done by biological means or by
using genetically modified plants or non-toxigenic strains of Aspergillus to reduce aflatoxin contamination by competitive
inhibition (Mishra and Das, 2003; Pitt and Hocking, 2006). Dorner and Horn (2007) have reported that the presence of
non-toxigenic Aspergillus has significantly reduced the growth of toxigenic species of Aspergillus. In another study,
Flavobacterium aurantiacum has been found to have activity to degrade aflatoxins (Styriac and Concova, 2002).
Modified glucomannans, derivative of yeast cell wall, has also been found beneficial in binding to higher levels of
aflatoxins at a lower inclusion rate (Devegowda and Raju, 2000; Murthy and Devegowda, 2004). Recently, Saccharomyces
cerevisiae and lactic acid bacteria were found to be effective biological detoxifying agents (Shetty and Jespersen,
2006). Similarly, nutritional approaches can also modify the toxicity of aflatoxins in animal body. An increase in dietary
protein protects against adverse effects of the toxin (Richardson et al., 1987). 5% fat in diet can also better the feed
conversion efficiency in pigs and improves weight gain in birds (Coffey et al., 1989). Adding methionine to broiler diet
improved the weight gains and reduced the adverse effects of AFB1 by increasing liver glutathione levels (Beers et al.,
1992). Feeding of extra vitamins A and K could also decrease the detrimental effects of aflatoxins (Lindemann et al.,
1993; Verma et al., 2001). Antioxidant compounds like selenium, chlorophyll and its derivatives, and medicinal herbs

7
 a b c
Fig. 1: (a) In situ photograph showing pale , enlarged, fragile liver with rounded margine in aflatoxin treated bird.
(b) Section of liver showing swollen hepatocytes in aflatoxin treated bird (H & E stain X 100).
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(c) Section of liver showing severe fatty changes in hepatocytes and mononuclear cell infiltration in portal area in aflatoxin treated
bird (H & E stain X 400).
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Fig. 2: Cattle liver (Aflatoxicosis) vascular congestion, Fig. 3: Dog liver (Aflatoxicosis)- fatty changes, congestion,
haemorrhage, coagulative necrosis. H&E x 100. degeneration. H & E x 100.

Fig. 4: Section of bursa of Fabricius showing depletion of lymphocytes

in bursal follicles in aflatoxin treated bird (H & E stain X 100).

8
 and plant extracts also reduces the toxic effects of aflatoxins (Gregory and Edds, 1984; Galvano et al., 2001). Juglal
et al. (2002) reported the inhibition of the growth of Aspergillus with spice oils such as clove and cinnamon oil.

CONCLUSION
Aflatoxins are considered the primary cause of abnormal health status and productive performances in domestic
animals and birds. Aside to this, economic losses arising from aflatoxicoses are enormous. In domestic livestock,
aflatoxin causes nutritional interference, there by hampering growth with serious implications on reproductive
performances. Aflatoxins cause huge losses to poultry industry by increasing susceptibility to various infections and
drastically reducing the production potential of flocks. Aflatoxins present as residues in milk, meat, eggs and other
animal products are of much concern to the humans too, as these toxins are much stable to heat treatments. The
corner stone to successful mycotoxin reduction in feeds is the control of moisture and temperature, which has a direct
bearing on the extent of mould colonization. Suitable detoxifying agents and toxin binders have to be used in order to
make sure that the animals are receiving safer feed materials. Continual research focusing on detoxification or
decontamination of feeds should be promoted. The use of biological detoxification and nutritional interventions strategies
should also be given much preference. Long term strategies should include nationwide strengthening of feed safety
measures, ensuring that feeds are harvested and stored properly. Detection of the fungal growth and presence of toxin
in feed stuffs has to be strictly monitored, for which the use of new generation tools and amenities should be exploited.
Agricultural and public health personnel need to demonstrate improved methods of feed utilization in farm households
and discourage the use of aflatoxin contaminated feeds. Organizations at National and International level are to
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constantly monitor the risk aflatoxins poses to animal health and need to draft legal limits for their effective control and
prevention.
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