Mycotoxins z In the broad sense, toxic substance of fungal origin. z Usually defined as fungal metabolites Mycotoxins that are toxic to man and/or animals and are produced by molds growing on foodstuffs or in homes and work places. z Molds: Asexual fungi classified in “form phylum” Deuteromycota

A Few Words About Molds A Few Words About Molds

z Molds z Definition: Sporangium „ Fungi with septate mycelium where „ Sporangium: Spores sexual reproduction is unknown or rare. spore containing „ Asexual reproductive spore = conidium structure. (pl. conidia). „ Asexual spore not produced in a sporangium.

A Few Words About Molds A Few Words About Molds z Definition: z “Form Phylum” Deuteromycota erected to „ Conidium: Asexual classify these fungi. spore not z Sexual reproduction stage lost or rare. produced in a z When sexual stage discovered, usually sporangium. Ascomycota, less frequently „ Conidia borne on conidiophore Basidiomycota. specialized hyphae (conidiophore) in Penicillium example.

1 Sexual spores of Basidiomycota A Few Words About Molds and Ascomycota z After discovery species reclassified, i.e. moved from Deuteromycota to Ascomycota or Basidiomycota. z This has never happened!

Basidium & basidiospores; ascus and ascospores

Example of Deuteromycota Example of Deuteromycota z Aspergillus variecolor = Asexual stage z Emericella variecolor = Sexual stage classified in Deuteromycota classified in Ascomycota

Chains of globose conidia borne on a swollen vesicle. Fruiting bodies of Emericella contains asci & ascospores

Example of Deuteromycota Example of Deuteromycota z Emericella variecolor = Sexual stage classified in Ascomycota z Emericella variecolor and Aspergillus variecolor are same species! z Aspergillus variecolor name should have been eliminated and only Emericella Star-shaped ascospore Î variecolor should be correct name. z Species never has been reclassified! Ascus with z Why? Í ascospores within

2 Significance of Mycotoxins zMycotoxins can be cause of illnesses in workplace and home. „Contraction of illnesses due to Mycotoxins in Food mycotoxin on spores. „Has been major problem in urban areas, Thursday – Inhalation of in high rises since 1970’s. Mycotoxins on Spores „Complaints reached epidemic proportions during 1990’s zMycotoxins released into food products can cause illness or death when consumed.

Discovery of Mycotoxins Discovery of Mycotoxins zRelatively recent discovery: zRelatively recent discovery: „In early 1950’s, hundreds of wild pigs „Although published, (Burnside, et al, died due to what is believed to be 1957), it seemed like old news. Why? consumption of moldy corn. „It would not be until 1960 when „Researchers isolated number of fungi mycotoxin poisoning would be from moldy corn. demonstrated. Grew fungi on sterilized corn and fed them to pigs. One species, Aspergillus flavus made pigs ill.

Turkey-X Disease Turkey-X Disease zIn 1960, approximately 100,000 turkeys zFungus identified as source of toxicity and lesser number of domesticated was Aspergillus flavus. birds mysteriously died, in England. zChemist isolated mycotoxin and called it zInitially thought to be a virus, it was aflatoxin (from A.flavus + toxin). named the Turkey-X disease. zDamage caused. zCause of disease traced to Oil Cake „Sensitivity to aflatoxin varied Mills, Ltd. according to animals tested. „Oil cake feed for turkey toxic. „Small amounts of toxin consumed could „Specifically, peanut was contaminated caused damage to internal organs, with mold. especially liver. One effect was cancer.

3 Turkey-X Disease Turkey-X Disease zThat peanut was implicated was of great zThat peanut was implicated was of great concern to world. concern to world. „Within United States, Food and Drug „Internationally, United Nations Administration (FDA) concerned. International Children’s Emergency Fund (UNICEF) and other similar „Internationally, United Nations organization concerned (continued). International Children’s Emergency Fund (UNICEF) and other similar organization Peanut used to increase protein in concerned because of: diet.diet Kwashiorkor severe malnutrition zQuality control in United States ensured caused diet high in carbohydrate, low in poisoning would be minimized. protein. „Standards set for aflatoxin.

Turkey-X Disease FDA Levels for Aflatoxins (in part) zQuality control in United States ensured poisoning would be minimized. Commodities Levels (ppm) „Random samplings indicated that Peanut products intended for feed 300 aflatoxin in peanuts and peanut products of beef cattle seldom occurred. Peanut products intended for feed 200 „Aflatoxin poisoning did occur in third of swine = more 100 lbs world countries without such controls. Peanut products intended for 100 breeding beef cattle, swine or mature poultry Peanut products for usage unknown 20

About Aspergillus flavus About Aspergillus flavus

zAspergillus flavus is actually a zSome of the species, e.g., A. oryzae, species complex of eleven species. used in preparation of foods in orient (continued). zSome of the species, e.g., A. oryzae, „ used in preparation of foods in Commercial strains utilized in United orient. States, of A. flavus, tested indicated aflatoxins not produced. „Soy sauce and tofu are two examples „ commonly used in this country. In strains, from households or village industry, tested from Philippines, all „Were we slowly being poisoned by sample found to have aflatoxin. eating contaminated food products?

4 About Aspergillus flavus About Aspergillus flavus zIf grains are pre-invaded with other zResearch indicated conditions that induce fungi, A. flavus contamination will not aflatoxin formation very narrow. produce aflatoxin. zAflatoxin produced by A. flavus complex zDemonstrated at University of Minnesota. when growing alone on grain, i.e. pure „Aspergillus flavus and other species culture. grown on grains for purpose of feeding to experimental animals. zNormally, when occurring on grain, a „Ducklings, white rats and baby chicks number of species of fungi will be growing fed contaminated grains. with A. flavus. Other species normally will invade first. „No deaths occurred in animals! Animals gained weight.

About Aspergillus flavus About Aspergillus flavus zChristensen (1972) sampled black z When aflatoxin produced, amount is pepper from all over the world. variable according to substrate: „In dilution plating, ground pepper was „More aflatoxin produced when A. found to have mostly A. flavus, A. flavus grows on peanut than in soy ochraceus and A. versicolor, all in bean, wheat corn barley, oat, etc. species complex. „Climatic conditions also a factor. „An average of 52,000 colonies/grams/black pepper. „Different isolates of A. flavus will „If wheat had a few thousand, or produce different amounts of barley 10,000 colonies per gram, they aflatoxin. Some will not produce any. would be rejected for milling and beer making, respectively.

About Aspergillus flavus Other Mycotoxins zChristensen (1972) sampled black zOchratoxin pepper from all over the world „Produced by Aspergillus ochraceus (continued). complex, composed of 9 species, and „If grains were as heavily contaminated Penicillium viridicatum. as black pepper would have a musty „Like aflatoxin, low amounts can be odor and taste. Why not pepper? lethal, e.g. will kill white rats. „Strong taste and odor of pepper „Lower amounts that does not kill will conceals aflatoxin. True also of other cause severe liver damage. spices.

5 Other Mycotoxins Other Mycotoxins zSterigmatocystin zFumagillin „Produced by Aspergillus versicolor, „Produced by Aspergillus fumigatus. which is part of A. flavus complex. „Unusual in that it is a mycotoxin and „ One of species found in black pepper. antibiotic. „Known to cause deaths in calves fed with „Used as an amoebicide, to rid body of grains contaminated by this species. amoebae that are human pathogens. „Causes tumors in lung, liver and kidney. „Too much and you get rid of the „Contamination of barley, in malt house, patient too! in Scotland.

Other Mycotoxins Other Mycotoxins zThe genus Fusarium zEstimated that 10% of population „Produces a number of important contracted “disease”, which was mycotoxins. fatal. Fusarium graminearum produces zSymptoms: vomitoxin, a trichothecene. „Nausea, vomiting and diarrhea. Fusarium trincinctum produces „Hemorrhaging in many organs. Trichothecene (T-2), Effects first discovered in Russia, in „Low white blood cell count. 1944s(!), associated with „Bleeding from nose and throat. alimentary toxic aleukia (ATA).

Other Mycotoxins Other Mycotoxins

zSymptoms were observed prior to zIt would not be until 1965 that WWII, but did not reach epidemic symposium on mycotoxin would this proportions until end of WWII. story be told. zBelieved to be due to inadequate zDiscovery of aflatoxin in 1960 number of people harvesting fall generated interest in learning about grain. mycotoxin throughout world. zBecause grain was not harvested until following spring it was heavily contaminated with fungal growth.

6 Trichothecenes Yellow Rain zResponsible for large scale deaths zTrichothecene Mycotoxin greater than ATA of 1944. „U.S. Secretary of State, Alexander „Following WWII, when Russian Haig in 1981 soldiers returned home, there was no food other than contaminated grain. „Consumption of contaminated grain led to mycotoxicosis of as many as a million soldiers.

Yellow Rain Yellow Rain zVietnam and Laos mounted attacks on zTrichothecene Mycotoxin Hmung tribes people of Northern Laos in „Haig accused the Soviet Union of 1975. supplying trichothecene mycotoxins to „Reports of terrorist attacks with Communist regimes in Vietnam and variety of toxic agents dropped from Laos for use in counterinsurgency low flying aircrafts. warfare. „About 70% of eyewitnesses reported seeing oily yellow liquid making sound like rain when it fell = Yellow Rain.

Yellow Rain Yellow Rain zVietnam and Laos mounted attacks on zVietnam and Laos mounted attacks on Hmung tribes people of Northern Laos in Hmung tribes people of Northern Laos in 1975. 1975. „Exposure reportedly caused heavy „Incidents investigated by U.S. medical bleeding from the nose and gums, teams 4-6 weeks later. blindness, tremors, seizures, other „Unable to determine cause of illnesses neurological symptoms, and death. and deaths. „Similar incidents later reported in Cambodia in 1978.

7 Yellow Rain zIn 1981, U.S. Army toxicologist Dr. Yellow Rain z Sharon Watson recognized symptoms as News media became suspicious of being same as trichothecene mycotoxin accusations and investigated evidence. poisoning. „Subsequent samples later submitted „Found evidence in analysis of dead and also tested positive, but with living victims and plant samples. different concentrations. „Haig accused Soviet Union of supplying „U.S. Army report in 100s of samples trichothecene to Vietnam and Laos. detected no trichothecenes. „Reagan accused Soviets of violating „Government reported that there were 1925 Geneva Convention and 1972 150-200 reports of such incidents, but Biological Weapons Convention. few witnesses.

Yellow Rain Yellow Rain zNews media became suspicious of accusations and investigated evidence. zNews media became suspicious of „Investigative teams carried out accusations and investigated evidence. “improper forensic methods”. „Investigative teams conducted investigation of incidents with Researchers made intentions of “improper forensic methods”. research known to villagers. No control. Asked leading questions. Asked leading questions. Non-random selections of witnesses. „People with “symptoms” often were Samples to be analyzed collected by found to have ingested food tribes people. contaminated with Fusarium.

Yellow Rain Yellow Rain zAnother theory: zIndependent investigation from other countries occurred: „Yale entomologist and expert on „Most did not reveal results. Southeastern Asian bees concluded yellow rain was bee feces. „Britain, France and Canada results endorsed U.S. findings. „U.S. scientists said that toxin was incorporated into feces to aide in „Australia was very skeptical about dispersal. accusations after investigation. „Yale botanist claimed pollen concentration too high to keep them airborne.

8 Yellow Rain Yellow Rain zAnother theory: zConclusion: „Analysis of feces did not come up with „Reagan administration was accused of trichothecene. fabricating “yellow rain” controversy. „Many accounts from entomologists and „Used controversy to increase defense others on local bees defecating en spending budget. masse as a “cleansing flight” usually „Aftermath: lasting for several minutes. Harmless! Former Soviet scientist has testified „“Defecation” occurred at same time as that USSR was experimenting with attack and became associated with trichothecene as biological weapon as yellow rain? early as 1958.

More on Trichothecenes More on Trichothecenes zTrichothecene and Population of Europe: zTrichothecene and Population of Europe: „Population stagnation in Europe „Population stagnation in Europe Before 1750, in England, life Before 1750, population was in expectancy of member of British constant fluctuation. Peerage was about 36.7 years. Many mortality crisis occurred: Conditions worst among common folks disease, famine, natural disaster. A hundred years later, expectancy After each crises, individuals married had risen to 58.4 years and population younger and more children were borne of Europe almost doubled. and population stayed even.

More on Trichothecenes More on Trichothecenes zTrichothecene and Population of Europe: zTrichothecene and Population of Europe: „Population stagnation in Europe „Population stagnation in Europe Believed changes in people’s diet Mary Matossian, in Poisons of the led to increase. Past suggest that increase in fertility During 18th Century, various grain was responsible for population breads were main source of food. increase. Matossian is suggested people’s fertility affected due to consumption of contaminated food.

9 More on Trichothecenes zTrichothecene and Population of Europe: „Population stagnation in Europe It would not be until change of diet that this trend would end. Recall between 1750-1850 was also time Irish population doubled.

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