Clinical and patho-anatomical effects of mycotoxins in animals

CLINICAL and PATHO-ANATOMICAL EFFECTS of MYCOTOXINS in animals

R. K. Asrani and Rakesh Kumar Department of Veterinary Pathology, Dr G C Negi College of Veterinary and Animal Sciences, CSK Himachal Pradesh Agricultural University, Palampur, Himachal Pradesh, India

1

Mycotoxins are secondary harmful mold metabolites that produce significant detrimental health effects in human beings and animals1. These are low molecular weight compounds known to be harmful even at low concentrations2.

Approximately 25% of the crops, including cereal grains and nuts, are often presumed to be contaminated with fungus3.

The most frequently encountered harmful mycotoxins in foodstuffs and feed include aflatoxin B1 (AFB1), ochratoxin A (OTA), trichothecenes, HT-2 and T-2 toxins, fumonisin B1 (FB1), citrinin (CTN), zearalenone (ZEN) and ergot alkaloids.

A predominately marked distribution of fumonisins, zearalenone and deoxynivalenol (DON) is documented globally .

Moisture content (20-25%)

4

Cereal crops may become contaminated in

Environmental temperature (22-30ยบC) Composition of food items

the field or during harvesting, transport, processing or storage5, 6. The rate of contamination of crops with fungus is more frequently triggered by the rainy season7.

Relative air humidity (70-90%) Physical damage to cereals by pests pH

Factors facilitating the production of mycotoxins in contaminated products include8:

Presence of mold spores

2

Diseases in animals

BIOLOGICAL FACTORS

Aflatoxixosis (AFB1): liver damage, liver cancer

Susceptible crop

Zearalenone (ZEN): reproductive problems etc.

ENVIRONMENTAL FACTORS Humidity Temperature Moisture Physical damage

Promote fungal growth in crops/ silage/concentrate/ grass, etc.

MYCOTOXINS PRODUCTION

Contaminated feed/fodder intake by animals

Secretion in milk and milk products (AFM1), eggs, meat and meat products (sausages)

HARVESTING, STORAGE & PROCESSING Consumption by humans (Mycotoxicosis)

Improper moisture, maturity and temperature conditions

Figure 1. Mycotoxins production and their occurrence in the food chain.

Common routes of entry of

Mycotoxins are known to produce several

mycotoxins into the body are:

harmful effects in animals and human beings.

Classification of these toxins can be made on

Direct consumption of contaminated

the basis of toxicity13 and clinical symptoms

products of plant origin (cereals, nuts,

related to the organs damaged14.

bread etc.) and products obtained from animals (meat and meat products, milk,

Highly toxic (lethal at 1-10 mg/Kg body weight) Trichothecenes Aflatoxin B1 Citreoviridin

offal’s, fermented sausages etc.)9, 10.

Dermal contact and inhalation are not very common routes but can act as a potential mode of entry into the body11.

Harmful toxic effects of mycotoxins depend on11,12:

Severely toxic (fatal even < 1 mg/ Kg body weight) Rubratoxin B Cyclochilorotine

MYCOTOXINS

Type of mycotoxin Dose introduced into the body Duration of exposure to the mycotoxins

Figure 2. Classification of mycotoxins on the basis of toxicity.

All other Mycotoxins (toxic > 10 mg/Kg body weight)

3

Heapatotoxicity (aflatoxins, rubratoxins, fumonisins, cyclochlorotine)

Nephrotoxicity (ochratoxins, citrinin, quinones, viomelein, xanthomegnin)

Figure 3. Classification of mycotoxins on the basis of clinical manifestations.

Neurotoxicity (fumonisin, patulin, citreoviridin)

MYCOTOXINS Estrogenicity (zearalenone)

Photosensitivity (sporedesmins)

Cytotoxicity (trichothecenes)

Immunotoxicity (ochratoxins, trichothecenes)

Table 1. Summary of different Mycotoxins with their toxic effects

Name of the fungus

Mycotoxins

Acronyms

Common sources

Target organs

Pathological change/Disease condition

Species susceptibility

Mode of action

References

Aspergillus ochraceus, A. verrucosum, Penicillium nordicum Aspergillus flavus, A. parasiticus

Ochratoxin

OTA

Kidneys, liver

Renal damage and cancer, hepatotoxicity

Swine, poultry, quail, human beings

Protein synthesis inhibition, nucleic acid damage and lipid peroxidation

15, 16, 17

Aflatoxins

AFB1, AFG1, AFB2, AFG2, AFM1

Liver, gastrointestinal system

Liver damage, hepatocellular carcinoma (HCC)

Pigs, human, dogs, cats, poultry

DNA adducts formation, mutations, inhibition of protein synthesis

18, 19

Trichothecium spp., Stachybotrys sp., Fusarium sp.

Trichothecenes

T-2, DON

Coffee, cereal grains, peanuts, dried fruits, cocoa, wine, spices, grape juice Sorghum, soybeans, nuts, rice, corn, cotton seed, cocoa beans, barley, dried fruits, crude vegetable oil Rye, wheat, barley, millet, oats

Gastrointestinal system and skin

Skin and gastrointestinal disorders

Horse, pigs, poultry, cattle, human beings

20, 21, 22, 23, 24, 25

Fusarium verticillioides, F. avenaceum, F. Tricinctum Fusarium graminearum, F culmorum Fusarium cerealis, F. roseum, F. incarnatum Fusarium proliferatum, F. verticillioides

Moniliformin

MON

Wheat,maize, rice,oats

Heart

Heart problems, depression

Birds

Inhibition of mitochondrial translation, inhibition of protein synthesis, DNA fragmentation Inhibition of thiamine pyrophosphatase pyruvate dehydrogenase

Zearalenone

ZEN

Wheat and maize bran

Reproductive system

Abortions,estrus defects, malformation of genital organs

Pigs, bovines, human beings

Binding with estrogen receptors, blocking the secretion of steroid hormones and suppressing estrogenic responses

28

Fumonisins

FB1, FB2

Silage, corn, wheat, barley, rice

Brain, lungs, esophagus

Swine, human beings, horses

Ergocristine, ergotamine, ergocryptine, ergometrine

Ergot alkaloids

Rye, wheat, triticale, barley, millet, oats

Smooth muscle, nervous system

Inhibition of sphingolipid biosynthesis, inhibition of protein synthesis in eukaryotic cells Partial agonist and weak antagonist in smooth muscles of the body, including blood vessels, and antagonist in central nervous system

29

Claviceps purpurea

Pulmonary oedema in pigs, Leucoencephalomalacia in equines Dry gangrene, abortion, hallucinations

Cattle, horse, sheep, poultry, human beings

26, 27

30, 31, 32

4

Effects of AFLATOXINS exposure Episodes of aflatoxicosis are

The order of severity of the mutagenic, immunosuppressive

associated with the production

and carcinogenic effects of aflatoxins is:

of aflatoxins by common fungal species such as Aspergillus

flavus and A. parasiticus in contaminated food products33.

AFB1> AFG1> AFB2>AFG233 AFB1 is predicted to exhibit developmental defects along with immune system dysfynction38.

In 1960, in the UK, the first report of mortality caused by aflatoxins-contaminated groundnut meal in turkeys

Hepatotoxicity

Teratogenicity

Immunotoxicity

Mutagenicity

Carcinogenicity

AFB1

and poultry was reported34.

The list of aflatoxins produced by

Figure 4. Harmful effects of AFB1

several fungal species includes AFB1, AFB2, AFG1, AFG2 and AFM1.

Species susceptibility to aflatoxins

Among all known aflatoxins, AFB1 is the most common and potent35.

All animal species are sensitive to aflatoxicosis, but outbreaks are usually encountered among pigs, cattle and sheep39.

Aflatoxins are very stable and are rarely destroyed after

The significant economic losses, including decline in growth rate

processing36. Additionally,

and productivity, are usually reported in farm animals depending

residues of aflatoxins

on individual susceptibility and the targeted species40, 41, 42.

are also reported to be excreted in milk, milk

Chronic exposure to AFB1 in farm animals can lead to various

products, meat and eggs33.

ailments, including liver dysfunction, compromised immune status and susceptibility to several diseases43,44,45,46,47,48.

AFB1 is well recognized for its hepatotoxic, teratogenic, immunotoxic and mutagenic

Some of the animal species, such as monkeys, chickens and

potential and is classified as group

mice have been found to be resistant to AFB149, whereas cattle,

1 carcinogen by International

horses and sheep are quite prone to AFB1-induced toxicity.

Agency for Cancer Research (IARC)37, as it causes hepatocellular

Younger animals have proven to be more

carcinoma in human beings.

susceptible than adult and older animals50.

Among aquatic animals, trout have been observed to be the most sensitive to AFB1 toxicity51.

Among poultry, the order of sensitivity is: ducks > turkeys> Japanese quail> chickens

.

52

5

AFB1

Metabolized by cytochrome P450 (CYP) in the liver to AFB1-8,9-exo-epoxide (AFBO), AFM1, aflatoxicol (AFL), AFB2a, AFQ1, AFP153

AFB1-8,9-exo-epoxide (toxic derivative and electrophilic in nature)

Binding with guanine residues of DNA and RNA in hepatocytes

Adducts induce mutations in DNA and inhibit DNA transcription and RNA translation54,55

Figure 5. Flow chart indicating mode of action of aflatoxin B1

Abdominal pain, vomiting and oedema can be observed in acute stages, whereas development of hepatocellular carcinoma is evident in later stages56.

AFB1 toxicity in ruminants leads to: Decline in ruminal motility Decline in the cellular digestion and fatty acid production Decline in feed efficiency and is secreted in milk as AFM1 after 12 h of consumption.

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Aflatoxin M1

Aflatoxin M1 (AFM1) is a group 1 carcinogen

(IARC) formed through CYP1A2-dependent hydroxylation microbial biotransformation from AFB1. The nuclear adducts are formed and secreted in milk and urine.

Aflatoxin B1 contaminated feed ingested by animals

Microbial biotransformation (CYP1A2-dependent hydroxylation) to AFM1

DNA adducts

Excretion in milk (DAIRY PRODUCTS) and urine Conjugation with glucuronic acid and excreted in bile

The concentration of AFM1 in milk is influenced by several factors, such as duration of lactation and the milk yield of the animal57.

Table 2. Permissible limits of aflatoxins consumption58, 59.

Agency

Maximum permissible limit of aflatoxins

AFM1 in milk as per US Food and Drug Administration (FDA)

0.5 μg/kg

Maximum permissible limit of AFM1 in milk and dairy products as per European Commission (EC)

50 ng/kg

Maximum permissible limit of AFB1 in dairy feed (FDA)

20 μg/kg

Health risk to infants and the human population

*An average intake of aflatoxins in human beings ranges between 10-200 ng/kg/day

Figure 6. Aflatoxin M1 in the food chain

Table 3. Clinical and patho-anatomical effects of aflatoxins.

Species

Clinical symptoms

Pathological changes

References

Humans

High fever, vomiting, tremors, hypoglycemia, coma and dark colored urine.

Elevated levels of AST, ALT, ALP, creatinine, catalase, malondialdehyde (MDA) and declined values of total proteins, magnesium and reduced glutathione.

Depression, anorexia, fever and ruminal contractions in a study conducted by Elgioushy et al.61.

Acute aflatoxicosis in Hereford calves after the consumption of peanut hay containing 2230 µg AFB1/kg led to symptoms including icterus, photosensitization, diarrhoea, depression and anorexia by a study conducted by McKenzie et al.62.

More sensitive to aflatoxicosis Contaminated feed consumption leads to reduced feed intake, declined growth and production status64.

Cerebral oedema, hemorrhages, fatty degeneration in liver and kidneys, encephalopathy, cirrhosis and hepatocellular carcinoma (HCC).

Liver enlargement, distended gall bladder, congested intestine with congestion of the kidneys61.

Proliferation of connective tissue involving portal triads in chronic toxicity of feed contaminated with aflatoxins63.

33, 60

Pregnant sows: distorted hepatic architecture, hemorrhages, distended sinusoids, cystic spaces in the liver, lymphoid depletion in lymph nodes and spleen65.

Hyperacute cases: hepatic necrosis and hemorrhages.

Acute toxicity cases: cellular infiltration, swollen hepatocytes with cholestasis.

Subacute toxicity cases: vacuolar degeneration, cholestasis with profound bile duct hyperplasia66.

Hydropic degeneration in the hepatocytes, necrotic changes, hyperemia, sinusoidal contraction with accumulation of ceroid pigments have been observed in macrophages from Merino rams treated with aflatoxins at the dose of 250 µg/day68.

Hemorrhagic spots in muscles, atrophied spleen; enlarged, paler, fatty liver with hemorrhagic areas; distended gall bladder, nephropathy and thickened crop and proventricular mucosa.

Microscopic fatty changes in hepatocytes, acinar arrangement of hepatocytes, lymphocytes and heterophilic aggregates with multiple areas of necrosis, hyperplastic changes in crop and proventricular mucosa70,71.

64, 65, 66

Cattle

Swine

Sheep

Decreased erythrocyte and leucocyte count with decreased values of hemoglobin and packed cell volume before initiation of clinical manifestations67.

Poultry

Feeding aflatoxins at a rate of 3.5 mg/kg of feed resulted in a marked decrease in body weight and growth performance with increased kidneys and liver weight69.

61, 62, 63

67, 68

69, 70, 71

7

Image 1. Gross pathological alterations associated with AFB1. Liver of a rabbit showing chronic hepatitis along with tumorous growth.

Image 2. Photomicrographs of pathological alterations associated with AFB1

a

b

c

d

a. Liver showing diffuse hemorrhages along with necrotic area in the hepatocytes along with hemosiderin deposition (H&E*66). b. Liver showing swollen hepatocytes with hydropic changes (H&E*66). c. Photomicrograph of liver showing portal fibrosis with bile duct hyperplasia (H&E*33). d. Liver showing peripheral shifting of nucleus giving a signet ring appearance indicating fatty changes in hepatocytes (H&E*66).

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Effects of OCHRATOXIN A exposure Aspergillus ochraceus, Auplopus

This mycotoxin was first reported

Pigs and poultry more

carbonarius and Penicillium

in contaminated cornmeal

sensitive to OTA

verrucosum are the most common

it is considered to be the most

fungal species associated with

common and potent mycotoxin

the production of ochratoxins

produced by these fungi73.

72

and

induced toxicity.

Ruminants are usually resistant, as OTA is degraded

in contaminated grains, raw and cooked food items and beverages

OTA is readily known for its

by ruminal microflora to

(coffee, beans, and wine).

nephrotoxic, carcinogenic,

less toxic metabolites

immunosuppressive, teratogenic

such as OTAÎą78.

Aspergillus ochraceus and

and genotoxicity in animals74,75,76.

Penicillium verrucosum are the

Additionally it has been found

Some researchers have shown

most potent moulds responsible

to produce hepatocellular

the release of OTA in breast

for the production of Ochratoxin

carcinoma as well, apart from

milk, which means it can act as

A (OTA) in tropical and

the nephrotoxic properties, in

a potent threat to the newborns

temperate regions, respectively.

a dose dependent manner .

through breastfeeding79.

77

Intake of OTA contaminated food items

Binding of OTA to blood albumin

Proximal convoluted tubule (PCT) (Target site)

Organic anion transporter (OAT) 1 and 3 help in the absorption of OTA in the interstitium and OAT 4 in the tubular lumen

Production of reactive metabolites that form adducts after reacting with DNA Alteration of the transmembrane potential of the mitochondrial membrane, causing the release of cytochrome c and apoptosis Inhibition of protein synthesis by competing with Phe-tRNA synthase

Figure 7. Flow chart indicating the mechanism of action of OTA in kidney tubular cells80 81 82.

9

Table 4. Tolerable limits of OTA. Agencies

Tolerable limits of OTA

Joint Expert Committee on Food Additives (JECFA)84

112 ng/kg body weight/week

Maximum limit of OTA (As per EU)85

3 Îźg/kg in processed cereal

European Food Safety Authority83

17 ng/kg body weight/day

*In one of the studies in Italy from Capei et al.83 has documented an 8% contamination of OTA in breakfast cereals and 50% contamination in sweet snacks with a contamination limit ranging between 2.9 â&#x20AC;&#x201C; 8.6%. A daily intake of OTA at the dose of 1 mg/Kg body weight for 5-6 days can be harmful.

Table 5. Clinical and patho-anatomicaleffects of ochratoxins.

Species

Clinical symptoms

Pathological changes

Referencias

Humans

Weakness, brown discoloration of the skin and lumbar pain.

Biochemical parameters such as glucose, gamma-glutamyl transferase and leucine aminopeptidase increase in urine, along with proteinuria with RBCS and WBCs in urine86.

86, 87, 88, 89, 90, 91

Swine

Bulgarian and Danish porcine nephropathy, reduced feed intake and weight gain92.

Residues can be transported to human beings through pork or offals93.

Poultry, quail and rats

In poultry, they cause a decrease in egg production, decreased FCR, immunosuppression, developmental abnormalities, reduced feed consumption with an increased water intake49.

Acute renal failure , Balkan endemic nephropathy (BEN), Tunisian nephropathy88.

BEN is a tubule-interstitial renal disease that leads to contracted kidneys in later stages86, which may also be followed by renal tumours89.

Tubular and glomerular degenerations with fibrotic changes in interstitial tissue, necrosis, apoptosis and end stage kidney damage90,91.

Swollen, pale, firm kidneys with gross lesions of fibrosis Microscopically, degenerative changes with fibrosis in the kidneys of pigs from abattoirs during random sampling were consistent finding in post weaning multisystemic wasting syndrome (PMWS), nephropathy syndrome (PDNS) and ochratoxicosis94.

Diffuse tubular nephrosis and interstitial fibrosis95.

OTA is found to majorly affect mitochondria in PCT cells and also causes degranulation of the rough endoplasmic reticulum (RER).

The changes associated with damage of kidneys in quail include profound karyomegaly, cellular swelling, cytoplasmic vacoulations, margination of chromatin material96.

Changes such as bile duct hyperplasia, necrosis of liver cells, vacuolar degenerations, dilation of central veins and sinusoids, along with mononuclear cell (MNCs) infiltration has also been reported in the liver by a research concluded by Patial et al.97.

Abnormalities of the central nervous system (CNS) along with skeletal system defects were reported in rats when administered OTA during the gestation period98.

87

92, 93, 94, 95

49, 96, 97, 98

Image 3. Gross pathological alterations associated with OTA. a. Swollen and pale kidneys of Japanese quail (right) after administration of Ochratoxin A in diet in comparison to the kidneys on the left side b. Ruffled appearance of feathers in a Japanese quail after feeding Ochratoxin A.

a

b

10

Image 4. Photomicrograph of pathological alterations associated with OTA. Kidney showing fibrous tissue accumulation in the interstitial tissue causing atrophy of renal tubules in OTA toxicity (H&E*66).

Effects of FUMONISIN exposure Fumonisins are produced

Toxicity associated to fumonisins

Fumonisins are also reported

by fungal species such as

was firstly reported in 1980 as a

to cause leukoencephlomacia

Fusarium verticillioides and F. proliferatum, and they are frequently spotted on maize giving it a whitish appearance99.

cause of equine encephalomalacia

in horses, hepatocellular

(ELEM) and porcine pulmonary

carcinoma in rats and pulmonary

oedema (PPE) in the United States,

oedema in association with

and esophageal cancer in Africa.

hydrothorax in pigs103, whereas the IARC has also documented

The most common forms of

These mycotoxins cause

the carcinogenic potential of

fumonisins include fumonisin A and

neurotoxicity, hepatotoxicity,

fumonisins in human beings104.

fumonisin B (B1, B2, B3 and hydrolyzed

embryo toxicity and

B1), and among these fumonisin B1

nephrotoxicity in animals101,102.

is the most common and potent

.

100

As per JECFA, the maximum tolerable limit of FB on the basis of no-observable-effectlevel (NOEL) of 0.2 mg/kg The production of this

bw/day with a safety factor

mycotoxin is promoted when

100 is 2 Îźg/kg/day105.

moisture content is < 19%.

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Mechanisms of action of Fumonisins102: Competitive inhibition of the ceramide synthase enzyme Oxidative stress and endoplasmic reticulum stress

Figure 8. Mechanisms of inhibition of sphingolipid metabolism.

Autophagy modulation Alteration of DNA methylation

Competitive inhibition of the ceramide synthase enzyme Required for sphingolipid biosynthesis

Disruption of sphingolipid metabolism

In liver, kidney and brain tissue

Inhibition of the acylation of sphingosine and sphinguanine

Inhibition of ceramide synthase

Table 6. Clinical and patho-anatomical effects of fumonisins.

Species

Clinical symptoms

Pathological changes

Reference

Humans

Esophageal cancer106.

106, 107

Cattle

Optic nerve degeneration leading to blindness108.

Swine - Porcine pulmonary oedema (PPE)

Defective vision, staggering, drowsiness, weight loss, decreased feed intake, respiratory distress and cynosis110,111,112.

Horses - Equine leukoencephalomalacia (ELEM)/Moldy corn poisoning) Fish

Circling, head pressing, blindness, ataxia and depression110.

Liquefactive necrosis or softening of cerebral subcortical white matter with occasional areas of hemorrhages.

Histological examination shows eosinophilic and swollen astrocytes in white matter of brain.

Pregnant female mice treated with 2.5 or 10 mg/kg FB1 intraperitoneal injection show neural tube defects in fetuses107.

Liver and kidney damage have been reported in calves treated with 1 mg FB1/kg body weight for 7 days109.

Histological changes evidenced in cattle with blindness includes fibrosed septa, retinal degeneration, optic nerve degeneration and oedema of myelin108.

Grossly, it is characterized by the presence of fluid in the thoracic cavity and airways with widened interlobular septa.

Microscopic evaluation depicts widened interlobular septa with perivascular and peribronchiolar oedema along with MNCs infiltration.

The right ventricle of the heart and pulmonary artery shows hypertrophy.

Vacuolar changes and cellular swelling of hepatocytes is also evident111, 113.

Focal to multifocal areas of necrosis of white matter.

Degenerative changes in endothelial cells along with perivascular thrombosis.

Oedema formation in neural tissue with neutrophilic infiltrations114.

Birds

Decrease in body weight, increased serum biochemical markers in ducks116.

Diarrhoea, increased gizzard, proventriculus and liver weight in broilers117.

Nervous symptoms115.

Chronic exposure of one year old carp (Cyprinuscarpio. L) with a feed containing FB1 at the dose of 10 mg/kg body weight for 42 days showed nervous manifestations.Histological evaluation of brain tissue reflected degenerative changes, vacoulations, necrotic changes in the brain cells around periventricular area and capillaries115.

Liver of affected quail showed necrotic hepatitis along with infiltration of heterophils and macrophages.

Increased Kupffer cell activity, bile duct hyperplasia with increased granularity of cellular cytoplasm has also been evidenced118. Additionally, kidneys showed swelling of the tubular epithelial cells and glomerular tuft, obliteration of Bowmanâ&#x20AC;&#x2122;s capsule, apoptotic changes and elevated mitotic activity118.

Deshmukh et al.119 concluded progressive degenerative changes along with heterophilic infiltration in tubular epithelial cells and interstitial tissue of kidneys at the dose of 150 ppm for 21 days in quail.

Hepatic necrosis, bile duct hyperplasia, thymic atrophy and rickets in broiler chicks affected with FB1 has also been observed in a study conducted by Ledoux et al.117.

108, 109

110, 111, 112, 113

110, 114

115

116, 117, 118, 119

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Image 5. Gross pathological alterations associated with FB1. a. Enlarged liver of a Japanese quail after feeding Fumonisin (FB1) for 3 weeks at the dose of 300 ppm. b. Enlargement of liver (right side) with Fumonisin (FB1) toxicity in comparison to normal liver on the left side.

a

b

Image 6. Photomicrograph of pathological alterations associated with FB1. Liver of a Japanese quail reflecting necrotic changes along with heterophilic infiltration admixed with mononuclear cells after the administration of FB1 (H&E*330).

Effects of TRICHOTHECENES exposure Trichothecenes are toxic secondary

The main mycotoxins belonging to

metabolites produced by Fusarium

the trichothecene group include type

graminearum, Stachybotrys, Fusarium poae, Fusarium langsethiae, etc, often found contaminating wheat, maize, barley and oat kept in damp environmental conditions.

A (T-2) and type B toxins (DON), and their toxic potential is due to the presence of an epoxide ring122.

These toxic metabolites are quite resistant to processing and are only destroyed at temperatures above

Production of these

260ยบC for more than 30 min.

mycotoxins is often favored by ambient temperature (0-32oC) with humid conditions120, 121.

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Harmful effects and tolerable limits of trichothecenes

In pigs, cattle, broilers and rats, trichothecenes

According to the EU, the maximum limit for the

damage the liver and stomach123.

presence of DON in cattle feed is 5 mg/Kg feed, whereas it is around 1 mg/Kg feed for calves.

Therefore, trichothecenes toxicity in farm animals is often associated to symptoms such as vomiting,

In studies conducted by Ingalls129 and Cote et al.130

diarrhoea, anorexia, weight loss and death

no marked variation in the milk production was

.

124,125

Additionally, the malabsorption induced by

reported when DON is given at a rate of 14 mg/kg

trichothecenes in pigs, poultry and rats is often

for 3 weeks and 66 mg/kg for 5 days, respectively.

associated with necrosis of intestinal villi126,127.

Based on the presence of ester-ether bonds between C-4 and C-15 at C-12 we can divide trichothecenes into 2 types: macrocyclic and non-macrocyclic. The nonThe maximum tolerable limits of

macrocyclic trichothecenes are enlisted in Table 7.

DON in most parts of the world are limited to 0.75 mg/kg in human diets and 1-5 mg/kg in animal rations128.

Table 7. Classification of trichothecenes.

A* Non-macrocyclic trichothecenes B

T2 toxin Diacetoxyscirpenol (DAS) Neosolaniol Nivalenol Deoxynivalenol (DON) Fusarenon-X

*T2 and DAS are used as bioweapons and are mainly produced by Fusarium poae and Fusarium langsethiae.

14

Inhibition of protein synthesis after binding to the 60S subunit of ribosome, leading to the inhibition of peptidyltransferase and inhibition of the initiation, elongation or termination steps in protein synthesis

Oxidative stress mediated DNA damage and apoptosis

TRICHOTHECENES

Figure 9. Clinical and patho-anatomical effects of trichothecenes20, 21, 22, 23.

Inhibition of mitochondrial translation

Table 8. Clinical and patho-anatomical effects of trichothecenes.

Species

Clinical symptoms

Pathological changes

References

Humans

Type A trichothecenes can lead to alimentary toxic aleukia (ATA) in human beings causing severe leukopenia, vomiting and nervous symptoms131,132.

Production losses, altered reproductive potential, decline in liver function and immunosuppression is seen in dairy cattle fed with silage and cereal grains contaminated with trichothecenes133.

Calves affected with DON toxicity show icterus with altered liver enzymes134.

Growth performance and feed intake in finishing pigs is severely influenced

by DON toxicity138.

Skin rashes, necrotic stomatitis, hemorrhagic vaginitis and nervous system affections131.

131, 132

Postmortem evaluation of dead carcasses reveals congestion and hemorrhages in abomasum, splenomegaly and kidney damage.

Histological investigation often reflects degenerative changes, cholestasis, bile duct hyperplasia, steatosis and infiltration of mononuclear cells (MNCs) especially macrophages and lymphocytes134.

T-2 toxicity in cattle leads to absence of estrus, hemorrhagic and necrotic enteritis, decreased feed intake and reduced milk production135,136,137.

133, 134, 135, 136, 137

Piglets fed with DON (1.5-2.8 mg/kg feed for 4-5 weeks) were found to show gastrointestinal and hepatotoxic pathological alterations139, 140.

DON interferes with reproductive potential of sows as is speculated to produce harmful effects on the ovaries and follicles141.

Feed contaminated with T2 toxin in acute toxicity cases lead to myocarditis/cardiotoxicity, rumenitis with ulcerative abomasitis, anasarca, brain oedema and pancreatic necrosis142.

138, 139, 140, 141

Providing T-2 toxin contaminated feed (10-20ppm) in juvenile goats led to severe pathological alterations in the intestine and liver on ultra-structural evaluation.

Apoptotic changes are markedly prominent in mesenteric lymph nodes, proximal convoluted tubules (PCTs) and distal convoluted tubules (DCTs) of the kidneys, enterocytes in the intestine and spleen with significant up-regulation of pro-apoptotic proteins, HSPs and cytokines143.

The histological changes in the liver tissue of goats included centrilobular necrosis, sinusoidal congestion, peri-ductular connective tissue proliferation, bile duct hyperplasia and vacuolar degeneration143.

143

Cattle

Swine

Sheep

Goats

Chronic exposure is often followed by declined reproductive potential, gastroenteritis, weight loss, myocarditis and pus in the oral cavity142.

Growth retardation, lethargy, decrease platelet, Hb and total leucocyte count, decrease in the values of serum and tissue superoxide dismutase and catalase143.

142

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Effects of ZEARALENONE exposure The most common fungal species involved in the production of zearalenone (ZEN) include Fusarium

culmorum, F. cerealis and F. graminearum. This mycotoxin is commonly found in cereal grains in temperate regions with warm weather144, 145 and can remain stable at temperatures up to 150˚C146.

The highest production of ZEN is reported at 25˚C with 16% moisture content147,148.

Five major metabolites of ZEN include α-zearalenone (α-ZEN),

Pigs are speculated to be the most sensitive species for ZEN-induced

β-zearalenone (β-ZEN), α-zearalenol

reproductive disorders as compared to other animals157.

(α-ZAL), β-zearalenol (β-ZAL) and zearalenol (ZON), α-ZEN having the

About 80-85% of oral dose of ZEN is found

highest estrogenic activity

to be efficiently absorbed in pigs158.

.

149, 150

Zearalenone is responsible for causing ear rot in maize and head blight in wheat and barley151, with immunotoxic, genotoxic, hepatotoxic and hematotoxic effects in animals, as well as significant nephrotoxic potential

The concentration of ZEN and α-ZEN in follicular fluid of swine is 38.9 and 17.6 pg/ml, respectively159.

Very limited data is documented about the folliculogenesis in ovaries of domestic animals160, but ZEN shows affinity towards estrogen receptors in uterus, mammary gland, brain and bones, which reflects its estrogenic potential161.

with an ability to produce pituitary adenomas152,153,154,155. Additionally, ZEN is linked to reproductive disorders in animals and hyperestrogenic syndrome in human beings156.

Table 9. Tolerable limits of zearalenone (ZEN).

Tolerable daily intake (TDI) in humans162 Cereals151

20-200 μg/kg Processed cereals

75 μg/kg

Unprocessed cereals

100-200 μg/kg

Unprocessed cereal snacks

50 μg/kg

Cereal foods

20 μg/kg

Regulatory limits (China) in wheat/corn/flour163

60 μg/kg

16

Figure 10. Mechanism of action of ZEN

Oral intake of ZEN

Absorption through the gastrointestinal tract

Converted to active metabolites (α-ZAL, β-ZAL) with the help of 3α and 3β hydroxysteroid hydrogenase

Binding to estrogen receptors in uterus, mammary gland, brain and bones, so reflecting its estrogenic potential

ZEN blocks the secretion of steroid hormones and suppresses the estrogenic response in the preovulatory phase

Table 10. Clinical and patho-anatomical effects of Zearalenone.

Species

Clinical symptoms

Pathological changes

References

Humans

Hypoestrogen syndrome in human beings, acting as a stimulating factor for precocious puberty development in females164.

Genotoxicity effect on the lymphocytes due to the formation of DNA adducts167,168.

ZEN is excreted in the milk of cows fed with high doses of ZEN. Heifers fed with 99% pure ZEN at a rate of 250 mg/day showed a decline in the conception rate of 87-62%169.

Weaned gilts affected with ZEN toxicity usually reflect ovarian atrophy, vulvar hypertrophy without any significant effects in uterus and mammary glands170,171.

Hormone production and estrus cycle length is not altered in mares provided with oats contaminated with ZEN (2 mg/ Kg)160.

ZEN leads to a decline in serum progesterone and testosterone concentration, reduced sperm count, increased incidence of infertility and decreased conception rate in pigs, cows, rats and mice168,174.

Endometrial hyperplasia, mammary tumors, adenocarcinoma and proliferative changes in women165,166.

164, 165, 166

Irregular estrus, infertility, abortion, retention of placenta, mastitis and metritis.

167, 168, 169

Decreased fertility, abnormal estrus cycle, abortion, vulvovaginits and reduced litter size172.

170, 171, 172

Ovarian follicular atresia173.

160, 173

Cystic mammary glands, hepatopathy and nephropathy, uterine fibrosis, persistent estrus, sterility, squamous metaplasia and, hyperplasia of the endometrial glands175,176.

168, 174, 175, 176

Cattle

Pigs Horses Rodents

17

Effects of MONILIFORMIN exposure Fungal sources involved in the production of monilformin (MON) include Fusarium

moniliforme, F proliferatum, F. avenaceum, F. subglutinans, F. tricinctum and Pencillium melanoconidium177, 178, 179, 180.

Contaminated cereal grains and plants used for silage

Inhibition of thiamine pyrophosphatase enzymes in tricarboxylic acid cycle

preparation are the major source of production of this mycotoxin.

MON is cardiotoxic and hematotoxic , with acute toxicity 181

Altered oxidation of pyruvate and Îą-ketoglutarate

that is comparable to trichothecene toxicity (T2, HT-2)182, 183. Fatal outbreaks of MON are reported in animals, but experimental studies in birds and rats have shown potential pathological effects184,185,186.

Inhibition of synthesis of collagen type II and aggrecan causing catabolic effect on articular cartilages

Inhibition of pyruvate dehydrogenase

Figure 11. Mechanisms of action of MON187, 188.

18

Clinical and patho-anatomical effects of moniliformin

In birds and laboratory rodents, intestinal hemorrhages are seen in acute cases, whereas cardiac hemorrhages are typical lesions in sub-acute and chronic cases of MON toxicity189.

In one of the sub-acute toxicity studies conducted by Jonsson et

al.190 reflected intestinal hemorrhages with pulmonary congestion in rats without other specific lesions in other organs.

Cardiomyopathy depicted by necrotic and degenerative changes in the heart with hypertrophy of muscle fibers causing cardiac arrest in quail birds fed with MON at the dose of 100 ppm has also been documented in previous studies191.

Image 7. Gross pathological alterations associated with MON. Japanese quail showing rounding and dilation of heart (Right side) after feeding MON at the dose of 110- ppm for 3 weeks; Left side showing normal heart.

Image 8. Photomicrographs of pathological alterations associated MON. a. Heart of a Japanese quail showing hypertrophy of cardiac muscle fibers following MON administration (H& E*132). b. Glomerular tufts occupied by needle shaped uric acid crystals in MON toxicity (H&E*66).

a

b

19

MULTI-MYCOTOXIN toxicity

In field conditions, it is most common to find raw materials to be contaminated with one or more mycotoxins, with variations in the symptoms associated with exposure, as the combination of these toxins can involve different types of interactions, such as synergistic, additive or antagonistic effects as shown in Table 11.

Table 11. Combined toxic effects of various mycotoxins.

Mycotoxin

Combination and type of interaction

Toxic effects produced

Ochratoxin (OTA)

Citrinin + FB1 (additive and synergistic)

Cytotoxicity to mononuclear cells192

FB1 (additive and synergistic)

Nephrotoxicity, hepatotoxicity, genotoxicity and immunosuppression

ZEN (antagonistic)

Cytotoxicity161, 197

Citrinin (synergistic, antagonistic and additive)

194, 196

Trichothecenes (synergistic and additive)

Nephrotoxicity, immune organ depletion, gastrointestinal problems and fetal malformations194, 196 Nephrotoxicity, immunotoxicity and hepatotoxicity198, 199

AB1 (synergistic and antagonistic)

Nephrotoxicity, teratogenicity, hepatotoxicity and cardiotoxicity

196, 200

MON (Synergistic)

191, 201

ZEN (antagonistic and synergistic)

Cardiotoxicity, nephrotoxicity, hepatotoxicity, immunosuppression, respiratory distress191, 201 Cytotoxicity and immunostimulation202

Trichothecenes (antagonistic and synergistic)

203, 204

Neural tube defects, hepatotoxicity, esophageal cancer

203, 204

Trichothecenes + ZEA (synergistic, antagonistic and additive

Cytotoxicity, oxidative damage and blockade of synthesis of macromolecules204, 205

204, 205

Fumonisin B1 (FB1)

References 192 193, 194,

195, 196

196, 200

193, 194, 195, 196 161, 197

198, 199

202

*The combined harmful effects of different mycotoxins depend upon the absorption rate206

20

CONCLUSIONS Mycotoxins are very harmful

Although in many of the countries

In order to limit the production

metabolites known to contaminate

tolerable limits for various

of mycotoxins, several strategies

food items and are majorly

mycotoxins are standardized,

are proposed and followed time

implicated in several clinical

a wide range of developing

and again by various agencies and

and pathological impairments in

regions around the globe still

regulatory bodies. In the present

human beings and animals.

need a thorough establishment

scenario, to minimize the production

of such standards with a strict

of mycotoxins during processing of

follow-up to reduce the levels of

raw material and final food products

mycotoxins in the food chain.

for animal or human use the basic principles to be followed include:

It is of utmost concern to prevent fungal contamination of food Excessive levels of mycotoxins can cause health hazards to the animals directly and through animal products to human beings.

products by providing high quality crops or animal products with controlled storage, harvesting and distribution strategies.

Good Agricultural Practices (GAP) Good Manufacturing Practices (GMP) Hazard Analysis Critical Control Points System (HACCP)

Regular monitoring of food items, animal feed etc. by employing proper guidelines and safety standards definitely will help to limit the fungal contamination.

21

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