Mycotoxins

MycotoxinsDr. Babasaheb Nagurao Kumbhar

M.V.Sc (Veterinary Public Health)[email protected]

Mycotoxins (Myco = of fungal origin) • toxic substances produced by fungi (molds) growing on

crops/grains in the field or in storage.• Secondary metabolites (chemicals) of a fungus that

produce toxic results in another organism.• Unlike bacterial toxins, fungal toxins (mycoatoxins) are

not proteins and therefore are not usually detectable by the immune systems of humans and animals

• Lack of visible appearance of fungus does not negate presence of mycotoxins. Toxins can remain in the organism after fungus has been removed.

• Cytotoxic: disrupt cell structures such as membranes, and processes such as protein, DNA, and RNA synthesis.

• Can be heat stable, not destroyed by canning or other processes.

Most fungi do not produce Mycotoxins

• Many fungi are edible• Mushrooms are fungi• Moldy feeds may be

degraded without presence of mycotoxin, or may be unaffected in value.

• some material courtesy of Mark Diekman

How was the concept of Mycotoxins developed ?

• An outbreak of an unknown disease killed poultry birds in 1960s

• This was named Turkey X disease• After investigations it was finally traced to

mycotoxins in groundnut meal feed imported from Brazil

• The feed was shown to contain a compound that could cause cancer

• Today we know this mycotoxin by the name of AFLATOXIN – A CLASS I CARCINOGEN

Brief History of Mycotoxins

• affected humans for thousands of years.• Middle Ages had outbreaks of ergotism.• Only in last 30-40 years have scientists been

able to isolate specific toxins from their fungal source.

• Research ideas and methodologies, in this field, change frequently, and data from 20 years ago are considered questionable.

• Alimentary toxic aleukia-killed over I lakh people

• Stachybotryotoxicosis-killed thousands of horse

Information About Fungus

• Range from single cells to fruiting bodies that form molds, mushrooms, smuts, and yeasts.

• Absorb nutrients from living or deceased organisms, contain no chlorophyll.

• If multicellular, they have tubular filaments called hyphae that branch out.

• Reproduce using spores.

How many mycotoxins are there?

• Today 300 - 400 mycotoxins are known• Mycotoxins of human concern based on toxicity: Aflatoxin Deoxyniva-lenol (DON) or Vomitoxin Zearalenone Fumonisin T-2 toxin Ochratoxin A

MycotoxinsFactors causing variation in effects

• Species, breed• Age• Sex• Nutritional status• Other diseases• Other mycotoxins• Extent of exposure

Some mycotoxins are formed in the field, some in storage

Storage conditions that favor production of mycotoxins: Temperature (40 - 90o F ; 4 - 32o C) Relative Humidity (> 70%) Moisture (22-23% in grain) Oxygen (1-2%)

Common toxigenic fungi

Aspergillus

Penicillium

Stachybotrys

Fusarium

Mycotoxin Chain of Events

Modes of Spore Transmission• Airborne, wind or indoor ventilation systems.• Attachment to insects of birds, thus transmitted

from plant to plant, or animal to animal, etc.• Via transportation mechanisms such as trucks, crop

machinery, etc.

Route of infection Ingestion/skin contact/ in halation

Blood stream & lymphatic system

Inhibit protein synthesis

Damage macrophages system

Inhibit particle clearance of the lungs

Increase sensitivity to bacterial endotoxins

Mycotoxins in corn

• Aflatoxins

Tricothecenes

• Fumonisins

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Common Mycotoxins

• Cool, wet weatherFusarium Toxins

• Deoxynivalenol (DON or Vomitoxin)– Considered a “marker”

• T-2 toxin (T-2)• Zearalenone (F-2)• Fumonisin

• Hot, humid weather• Aflatoxin

Fungi Substrate Mycotoxin

Aspergillus flavus Maize, groundnut, oilseed, cotton seed

Aflatoxin

Aspergillus parasiticus Maize, groundnut, oilseed, cotton seed

Aflatoxin

Aspergillus nomius Maize, groundnut, oilseed, cotton seed

Aflatoxin

Aspergillus ochraceus Barkey wheat Ochratoxin

Aspergillus carbonerius Grapes wine coffee Ochratoxin

Fusarium oxysporum Wheat barley maize Fumonisins

Fusarium sp. Wheat barley maize T-2 toxin

Penicillium verrucosum Wheat barley maize Ochratoxin

Claviceps purpurea Rye Ergot alkaloids

Stachybotrys Hay Satratoxins

Toxicity of Mycotoxins

Acute ChronicMutagenicTeratogenic

Acute toxicity

• Deterioration of liver and kidney functions, leading to death in extreme cases

• Interference with protein synthesis• Skin sensitivity affected• Necrosis of skin• Immunodeficiency• Neurotoxins cause trembling in small

doses and brain damage or death in slightly higher doses

Chronic toxicity

• Induction of cancer (Liver, oesophagus)• Induction of tumors• Induction is never detected at the time of

ingestion and remains undetected till the disease is advanced

Mutagenic & Teratogenic toxicity

• Replication of DNA is affected producing mutagenic and teratogenic effects

Determination of mycotoxins

• Presumptive tests

• Biological assays

• Chemical analysis

Potential uses of mycotoxins• Medical application – • Trichothecenes are known to possess

antileukaemic activity; • zearalenone derivatives have been considered as potential treatment for menopausal syndrome in women;

• Agriculture application – derivatives of zearalenone as growth promoter in sheep and cattle; other have been considered for use as herbicides and insecticides.

Effects of Molds/Mycotoxins

Molds - Decrease digestibility (5%) - Decrease production (5-10%)

Mycotoxins – Impact on milk production, weight -- gain greater than molds Mimic other herd health problems

– Depressed immune system– Off-feed– Diarrhea– Hemorrhaging– Abortions– Reproduction issues

Health effects of mycotoxins

Acute and chronic effects on both humans and livestock

Mycotoxins are believed to be among the most potent known carcinogens

Effect range from immediate toxic responses & immunosupression to potential long-term teratogenic, estrogenic, & carcinogenic effects.

Possible health effects due to airborne exposure (exposure to airborne spores with mycotoxins)

Human mycotoxicosis

In 1967, 26 Taiwanese in a farming community became ill after eating contaminated rice; 3 children died.

Cause of death: Contaminated rice showed >200 µg aflatoxin B1/kg.

In 1974, an outbreak of hepatitis in Indiaaffected 400 people resulting in 100deaths;Cause of death: aflatoxins in corn ( >15

mg/kg)

Human mycotoxicosis

In 2004, one of the largest aflatoxicosis outbreak occurred in rural Kenya resulting in 317 cases and 125 deaths.

Cause of death: corn contaminated with 4,400 µg/kg of

aflatoxin B1, 220 times higher than Kenyan regulatory limit for food.

Mycotoxin Toxicity: Effect on Humans• Physiological and pathological changes• Food poisoning• Inhibition of protein synthesis & Alteration of capacity

of cells to proliferate• Increase of tryptophan in blood and brain (affects

appetite, muscular co-ordination and sleep• Nausea ,Vomiting• Headache• Abdominal pain, Diarrhoea Giddiness• Convulsions• Reproductive and mammary changes• Role in hormonal balance and breast cancer• Precocious pubertal changes in children• Breast enlargement in boys• Role in cancer• Immunosuppressor

Symptoms of Mycotoxicosis

1. Drugs and antibiotics are not effective in treatment.2. The symptoms can be traced to foodstuffs or feed.3. Testing of said foodstuffs or feed reveals fungal

contamination.4. The symptoms are not transmissable person to

person.5. The degree of toxicity is subject to persons age (more

often in very young and very old), sex ( more often in females than males)and nutritional status.

6. Outbreaks of symptoms appear seasonally.

Mycotoxin Effects on Animals• Feed refusal.• Impaired animal health, resulting in reduced

production of eggs, milk, weight gain, etc.• Metabolites are passed through the milk in

cheese, dry milk, and yogurt.• Disease.• Death in animals.

Some important mycotoxins

Fusarium plant pathogens in soil • corn, wheat, barley, beans, • Trichothecene toxins target the circulatory,

alimentary, skin, and nervous systems.

F. graminearum- WheatCauses scab damage to kernels and head blight.Produces deoxynivalenol (DON), also called vomitotoxin.

F. graminearum in Maize (Corn)• Produces the toxins: DON & zearalenone (ZEN),

and T-2 toxins.• Have damaging effects on plants, humans, and other animals with monogastric digestive

processes.

Zearalenone T-2 toxin

F. moniliforme-corn• Plant pathogen most associated with corn. Also found in

rice, sorghum, yams, hazelnuts, pecans, and cheeses.• Diseases “crazy horse disease” in horses, pulmonary edema in pigs, liver cancer in rats, bone malformation in chicks and pigs. esophageal cancer in humans.

• Other toxins produced include fusaric acid, fusarins, and fusariocins.

• Advisory levels are 5 ppm in animal feed.

DON and T-2 Toxin

• tricothecenes of wheat, grain, and barley.• necrosis and hemorrhage of the digestive tract,

decreased blood production in the bone and spleen, and changes to reproductive systems.

• In poultry, causes reduced egg production, beak lesions, and abnormal feathering

• Optimal temperature range is between 70 and 85 degrees Farenheit.

• Advisory level of DON is 1 ppm.

Alimentary Toxic Aleukia (ATA)• During World War II, in Soviet Union, corn grain left to

over-winter becomes contaminated with T-2 toxin.• Severe mycotoxicosis occurs.• burning in the mouth, esophagus, tongue & stomach.• Bone marrow formation is halted & anemia develops.• Hemorrhage of nose, gums, mouth and stomach occurs.

Zearalenone

Species : Fusarium roseum, F.graminearum, F. poae, F. culmorum

Food affected: corn, wheat, barley, oatsInteresting facts: • Zearalenone has estrogenic effects.

Zearalenone

Mimics the body’s production of estrogen. Lengthened or absent estrous cycle Causes feminization of male animals. Prepuberal gilts show enlarged, swollen vulva

as if in estrus Interrupted reproductive cycles in female swine Prolapse of the vulva possible Little or no effect on growth Pigs are especially sensitive, poultry and cows show little sensitivity.

Alternaria Toxins

• wheat, sorghum, and barley.• Also fruits and vegetables that can cause spoilage in

refrigeration.• Toxins include: alternariol, alternariol monomethyl

ether, altenuene, tenuazonic acid, and altertoxins.• Little is know of these toxins; but, toxic effects are

seen in rats, chicks, ducklings, and turkeys.

Claviceps Toxins

• Earliest recognized mycotoxicosis caused by C. purpurea, with ergot mold.

• Outbreaks have been reported since 857 A.D.

• Humans consumed bread baked with grain containing ergot spores, which produced lysergic acid diethylamide (LSD) symptoms and hallucinations.

Ergot Poisoning (St. Anthony’s Fire)

• Disease of barley, oats and wheat.•Grains are replaced by ergot sclerotia that contain toxins.•Main toxin is called ergotamine.•Named for the belief that a pilgrimage to the shrine of St. Anthony would alleviate the symptoms. Ergotamine

Species:

Claviceps purpurea, and Aspergillus and Penicillium

Affected foods :- rye, barley, wheat, and oats

Interesting facts: • First documented case of ergot poisoning was in 857. [5]• Ergot contains a chemical precursor to lysergic acid

(LSD).

Ergot Poisoning (Cont…)

•Animal symptoms:• Dry gangrene,• Internal bleeding,• Vomiting,• Constipation, •Diarrhea, and

•In pigs: abortion of fetuses.

Human symptoms: Gastrointestinal stress; convulsions; Fetal abortion; Extreme burning and Cold sensations in Fingers, hands, & feet; Gangrene of extremitie

Aspergillus Toxins• more than 100 species, 50 of which are

known to produce mycotoxins.• Aspergillus niger is used to make artificial

citric acid; one use is in soft drinks.• Miso, soy sauce, and sake use strains of A.

oryzae.

Cotton, peanut, maize spices, pistachoos

Aspergillus and Aflatoxin• Aflatoxicosis: caused by high doses in short

intervals or low doses in high intervals.• 1961, caused the deaths of over 100,000 turkey

poults: “Turkey X disease”.• Toxin was traced to contaminated Brazilian

peanut meal in poultry feed.• Grows best between 80-90 degrees Farenheit.• Damage to grain increases likelihood of fungal growth.

Aflatoxin1. Sources :Corn, peanuts2. Factor favoring production of aflatoxins

a. Temperature : 25-30 ๐cb. Grain moisture

highly toxic carcinogenic secondary metabolites produced by fungi namely:-

1. Aspergillus flavus

2. Aspergillus parasiticus

3. Aspergillus nomius

Types of Aflatoxins

• Naturally produced Aflatoxins –B1, B2, G1, G2

• They undergo modifications during cellular metabolism and processing of foods to produce several derivatives such as

M1, M2, P1, etc.

International limit 15mg per kg food

Aflatoxin B-1• most potent aflatoxin• Classified as Class I Human carcinogen• selectively targets human gene p53 described as ‘Guardian of the genome’• Definite link to cancer in animals.• Primarily attacks the liver, in cases of cirrhosis, necrosis,&

carcinomas with a secondary affect immune suppression. • Risk factor for neonatal jaundice, in areas of maternal

consumption.• Does not stay in the body for long periods of time, usually

excreted within 96 hours, in animals.• In milk, for human consumption, advisory level is .5 ppb.

AFLATOXIN EFFECTS

Inhibits protein synthesisPoor gainLiver damageSusceptibility to InfectionResidues / carcinogenicityReproduction in swine not primarily affected

Penicillium Toxins

• Large genus with over 150 species.• Discovered antibacterial properties within

genus, causing production of penicillin.• 100 species have mycotoxins.• Nine specific toxins affecting human health are

citreoviridin, citrinin, cyclopiazonic acid, ochratoxin A, patulin, penitrem A, PR toxin, Roquefortine C, and, Secalonic acid D.

Penicillium Toxins (Cont.)

• Separated into two groups: those that affect liver and kidneys, and those that are neurotoxic.

• Liver and kidney toxins are asymptomatic and cause overall animal debility.

• Neurotoxins cause visible trembling.

http://farma.qfb.umich.mx/images/penicillium.jpg

Ochratoxin A and Citrinin• Affects kidney function.• Causes Balkan nephropathy and Yellow Rice Fever in humans.• Chickens, turkeys, and ducklings are affected by ochratoxicosis,

causing poor weight gain, egg output, and poor shell quality.• Ochratoxin sources are peanuts, pecans, beans, dried fruit and

dried fish.• Citrinin sources are in wheat, rice, corn, and flour.• Citrinin is most associated with horses, pigs, dogs, and poultry.

Ocratoxin A Citrinin

Ochratoxin A

Species: P. Verrucosum and A. ochraceusFood affected: Cereals, coffee beans, and grapes.Interesting Facts: Ochratoxin can be transmitted from pork to humans by eating pork that is fed with contaminated food.

Cyclopiazonic Acid (CPA)

• Found in corn and peanuts in Georgia.• Chief species from Penicillium causes cheese

spoilage.• Causes fatty degeneration in liver and kidneys

in animals, chickens are very susceptible.• May act synergistically with aflatoxin.

CPA

Types of Tests for Mycotoxins Quick Test (Qualitative)

Immunoassays (Elisa tests) Thin Layer Chromatography (TLC)

Use: (Detect Specific Mycotoxin)

Confirmatory Tests (Quantitative) High Pressure Liquid Chromatography(HPLC)

Use: Determine level of mycotoxins Detect several mycotoxins

Managing Moldy/Mycotoxin Problems

Ensure adequate levels of vitamins (A,E,B-1) and minerals (Se,Cu,Zn,Mn).

Include aluminosilicate or bentonite in ration

Dilution is the solution. Reduce intake of contaminated feeds.

Eliminate highly contaminated feeds. Remove moldy layers of feed before feeding.

Quick Toxin ReviewOrgan System Affected Toxin(s)

Vascular AflatoxinDigestive Aflatoxin, T-2toxin,

VomitotoxinRespiratory Trichothecenes

Nervous Trichothecenes

Cutaneous Tricothecenes

Urinary Ochratoxin A, Citrinin

Reproductive Zearalenone, T-2 toxin

Immune Many

Future Fight Against Mycotoxins• Have farmers select strains resistant to

contamination.• Scientists hope to genetically engineer plants

resistant to fungal infection.• Use feed additives that sequester the toxins

and prevent absorption from the gastrointestinal tract.