Lecture11: Mycotoxins

Fungi that cause diseases are actively feeding/growing on our bodies. However, there is another means by which fungi can impact our health. When fungi grow on stored food material that we consume, they may produce harmful metabolites that diffuse into the food. It is believed that fungi evolved these metabolites as a means of protecting their food supply by inhibiting the growth of other fungi and to discourage animals from eating their food. These metabolites are referred to as mycotoxins, which literally means "fungus poisons". In the broad sense, mycotoxins are toxic substances of fungal origins. However, typically they are defined as fungal metabolites that are toxic to man and/or animals and are produced by molds growing on foodstuffs.

Fungi that are referred to as molds have a fungus body that is characterized by having mycelium that is septate, yeast or may be dimorphic. Normally, fungi with these characteristics would be classified in either the Ascomycota or Basidiomycota if their sexual stage are known. However, there are many species of such fungi that have either lost their ability to reproduce sexually or only rarely do so. Because of the absence of a sexual stage, a "form phylum" the Deuteromycota was erected to accommodate these fungi and classification within the phylum is based on their asexual stage. Reproduction occurs by asexual spores called conidia that are borne on modified hyphae called conidiophores. When sexual stages of molds are discovered, they are usually members of the Ascomycota and less often in the Basidiomycota. 

Because it is the mycotoxin that has caused food to become contaminated, the mold that produced the metabolite does not have to be present. If, for example, a fungus was growing in a silo where grain is stored, the environment may become unsuitable for the fungus and will die off. However, even though the fungus is no longer present, while it was growing on the grain, its mycotoxin may have poisoned the grains. So for those of you who are always looking to save a little money by buying cheese that has been contaminated with a fungus and cutting out the part where the fungus is growing, perhaps this is not such a good idea. It is possible that the fungus growing on your cheese has produced a mycotoxin that has diffused throughout the cheese, even though the fungus itself has not yet grown over the entire surface of the cheese. The poisoning by mycotoxin is referred to as mycotoxicoses. The knowledge that mycotoxicoses is the result of fungal actions was a relatively, recent discovery. This is understandable since illnesses in this case is due to consumption of mycotoxins that has been released by the fungus and is not directly caused by the fungus. So demonstrating that this has occurred was not an easy task.

Early Attempts to Demonstrate the Existence of Mycotoxin

The existence of mycotoxins was not documented until 1960. However, just as in the case of diseases, the concept that moldy food could lead to illness in people or domestic animals was long suspected before their existence was demonstrated by science. It is a greater problem, presently, than it was in the distant past. Long ago, before there was adequate means of long term storage for perishable goods, food was normally consumed a short time after it was acquired, but as the world has become more industrialized and technological advanced, storage of food has become more of an issue. Food is now commonly stored for long periods of time, giving fungi a greater opportunity to contaminate our food.

Before 1900, in Italy, researchers believed consumption of moldy corn by children led to the development of illness (Christensen, 1975). Some experiments, done at that time, included the isolation, and growth of the suspected fungus in pure culture, and isolation of toxic compounds from the fungus that the researchers believed to be the cause of the illness. However, since the compound was not identified and was not actually isolated from the moldy corn, it could not be concluded that this compound was the cause of the illness or that the compound in question was even present on the moldy corn. Nevertheless, it appeared that there was a correlation between the illness and consumption of moldy corn, but this did not eliminate the possibility that it was the fungus, itself, that caused the disease, which most people believed to be the case. It was also possible that there were other reasons for the illnesses that were observed.

Burnside, et al (1957) studied an extensive outbreak of moldy corn disease in the southeastern United States in the early 1950's where hundreds of wild pigs foraging in cultivated corn fields became ill, and many died. Teams of veterinarians and mycologists collaborated to determine the cause of the deaths of these pigs. They isolated a number of different fungi from the moldy corn and inoculated each fungus on moist corn that had been sterilized and then fed them to pigs. The consumption of corn inoculated with Aspergillus flavus caused outward signs and inward lesions found in other cases of the so-called moldy corn disease. However, since there was no toxin(s) isolated, there was little attention paid to this paper since it still seemed like old news, i.e. domestic animals poisoned by eating moldy corn. In hindsight, it is surprising that this research was published at all.

It would not be until 1960, when approximately 100,000 turkeys and a lesser number of other domestic birds died in England, causing losses of approximately several hundred thousand dollars, before the first mycotoxin would be isolated and identified. Initially, the disease was thought to be caused by a virus and the syndrome was named "turkey-X disease". The "X" here indicated that the cause of the disease was unknown. However, with a great deal of detective work, on the part of the researchers, soon the cause of the disease was traced to feed that was produced by Oil Cake Mills, Ltd. (research always seems to get done more quickly and receive more priority when loss of large sums of money is involved). The oil cake feed was composed mostly of peanuts. However, it seemed unlikely that the peanut meal itself was toxic, since peanut meal had long been used as a feed ingredient and was known to be an excellent source of protein. Thus, it was reasoned that something must have been added to the peanut meal to make it toxic, and one possibility that was investigated was that peanuts had been made toxic by fungi growing in them. From their isolations, the investigators identified Aspergillus flavus, the same fungus that was isolated by Burnside and his research teams. The isolated fungus was again inoculated into the feed and fed to the turkeys. Shortly after feeding, the turkeys died with external signs and internal lesions identical to those observed in the birds that had previously died in the field.

Unlike Burnside, however, chemists were also employed in this investigation, and they were able to isolate and identify the toxin from the oil cake feed. The mycotoxin isolated was named aflatoxin, the "a" from Aspergillus and "fla" from flavus. Feeding test of food containing aflatoxin, with various laboratory animals, demonstrated that to varying degrees, all animals tested were sensitive to aflatoxin. Even consumption of extremely small amounts of aflatoxin damaged various internal organs and could induce development of cancer to the liver.

This was of great concern to the Food and Drug Administration (FDA). There was great concerned domestically since peanuts and peanut products were/are of economic importance. It was also of international significance, since peanuts at that time was being lauded as an excellent source of protein, for developing countries, by UNICEF (United Nations International Children's Emergency Fund) and other such organizations. Deficiency in protein often results in "kwashiorkor".

Kwashiorkor (kwä´shê-ôr´kôr´), protein deficiency disorder of children, prevalent in overpopulated parts of the world where the diet consists mainly of starchy vegetables, particularly Africa, Central and South America, and S Asia. Such a diet is deficient in certain amino acids, which make up proteins vital for growth. Depending on the extent, onset, and duration of the deficiency, manifestations include skin changes, edema, severely bloated abdomen, diarrhea, and generally retarded development. From the Concise Columbia Encyclopedia is licensed from Columbia University Press. Copyright © 1995 by Columbia University Press. All rights reserved.

Other characteristics include anemia, depigmentation of the skin, and loss of hair or change in hair color. Usually, occurring in children, shortly after weaning. Peanut products were developed in various forms, especially in tropical and subtropical countries, for general distribution. Were these people now exchanging kwashiorkor for potential risk of liver damage and cancer from consuming peanut products tainted with aflatoxins? There was a great deal at stake and answers were needed immediately. In the United States, as soon as awareness of aflatoxins surfaced in the 1960s, programs were established by peanut growers, aided by concentrated research, to reduce the possibility of aflatoxins occurring in edible peanuts and peanut products. Maximum acceptable aflatoxin levels were established for peanut products and random sampling was carried out by the FDA approved labs. Only occasional batches of peanut products have been found found to contain aflatoxins. What has been discovered have almost never been found in sufficient amount to be of any real concern. Testing for imported peanuts also is also required by the FDA. However, where quality control is absent, unsafe levels of aflatoxin is probably common, e.g. peanuts(sic) butter prepared by street vendors in Sudan (Elshafie, et al. 2011).

The Fungus

Aspergillus flavus is actually not a single species, but a "species complex", made up of 23 species that difficult to separate because of their overlapping morphological characteristics that are used to define the individual species (Hedayati, et al. 2007). These species are known to occur in many kinds of plant materials, including stored grains. One of the species in the complex, A. oryzae has long been used in the Orient to prepare various kinds of food products, such as sake, tofu and soy sauce, which in turn is used in the United States. However, it is also known to cause mycoses such as meningitis, paranasal sinusitis and pulmonary infections (Hedayati, et al. 2007). Were aflatoxins present in these products as well? This and other questions concerning this species complex needed to be answered and research began to take place at a rapid pace and continues to do so. The number of papers published that have to do with aflatoxins number in the hundreds annually.

What was determined in early research of aflatoxins is that the conditions which allows for growth of A. flavus and aflatoxins is very narrow. Aspergillus flavus seldom invades stored grains alone, i.e. as a pure culture. Various other species of fungi will normally grow on a substrate prior to invasion by A. flavus, e.g. A. glaucus and Candida pseudotropicalis. In a preinvaded substrate, regardless of how dense the A. flavus invasion may be, aflatoxin apparently does not form. This was demonstrated by Christensen (1975) where A. flavus as well as other fungi were grown on grains at moisture content and temperature range that were optimal for aflatoxin production. Although, there was a very dense mycelial growth of A. flavus, the contaminated grain that was fed to the various kinds of experimental animals, ducklings, white rats and baby chicks, for as long as eight months, there was not a single case of death from consumption of the feed (Christensen, 1975). In fact, weight gain was the same as that of those animals fed on fungus free grain. Thus, the amount of mycelial growth that occurs in animal feed, with several species of fungi involved, even if one is known to be an aflatoxin producer, was apparently safe to consume. In order for aflatoxin formation to occur in say a storage bin full of peanuts, A. flavus must be growing alone and the peanuts cannot have been previously or simultaneously invaded by other fungi, an occurrence that is rare. In the case of the Turkey-X disease, the peanuts that were responsible for the aflatoxin poisoning were from South America, where the process used to harvest and dry the peanuts was responsible for providing an environment that allowed for formation of aflatoxin. Aspergillus flavus does not normally contaminate grains and other crops while they are still in the field. It is only after the grains are harvested and stored does A. flavus, as well as other so-called "storage fungi" that have a low moisture requirement, can the grain be invaded. Although conditions favorable for growth of the A. flavus and production of aflatoxin is narrow, the fungus is common and widespread in nature. It can be found growing on various decaying vegetation where it may heat up the substrate to as high as 113-122°F as it consumes the material.

The amount of aflatoxin formed differs as to the substrate on which it is growing. Although the mycelial mass may be the same in each substrate, the aflatoxin produced would be far greater in peanuts than in say soybeans, where relatively very little would be produced. Other seeds of cereal crops, wheat, corn, barley, oats and sorghum are also generally of low-aflatoxin-risk. Weather and climate were also contributing factors. Finally, the amount of toxin produced will vary with the isolate of A. flavus. That is different sources of A. flavus will produce different amounts of aflatoxins. Some isolates of A. flavus may not even form aflatoxin.

Strange as this may sound, in some cultures, fungi are encouraged to grow on certain foods in order to give them the desired taste. For example, the Bantu tribes in Africa prefer the sour flavor of partly spoiled corn to that of fresh corn and fungus is purposely allowed to grow on the corn for this reason (Christensen, 1975). However, this may only be a coincident, but they also have a very high incident of primary liver cancer.

How much aflatoxin is too much?

Christensen (1975), over a period of several years, examined 100 different samples of black pepper from all over the world. In dilution cultures of these samples, the number of fungus colonies in whole or ground black pepper averaged 52,000 per gram/black pepper and the upper range was over half a million per gram. These colonies were mostly of A. flavus, A. ochraceus and A. versicolor. All three species are known to be aflatoxin producers. 

How heavily contaminated is 52,000 to 500,000 colonies of fungi, per gram? Lets make a comparison for what is acceptable levels of fungal colonies isolated in other food products at the time Christensen published his results. Wheat, for example, that is intended for milling into flour seldom contains no more than a few thousand colonies of fungi per gram of grain. If barley has as many as 10,000 colonies of the same kind of fungi per gram as in black pepper, it would be rejected for malting in beer making. If breakfast cereals or bread were as contaminated as black peppers, they would have so musty an odor and taste that they would be too revolting to eat. Christensen (1975) commented that the natural spicy odor and flavor of black, as well as white pepper are potent enough to conceal the taste and odor of these fungi. However, he did not demonstrate the presence of aflatoxin. Afterall,  concealment of odor and taste cannot free contaminated pepper of aflatoxin, if it is present. Madhyastha and Bhat (1984) carrying out a similar experiment, with black and white peppers, using Aspergillus parasiticus, another aflatoxin producing species, concluded that piperine and pepper oil, in pepper, inhibited mycelial growth, but more importantly also inhibited aflatoxin formation. 

What about the processed food prepared with A. flavus? According to Christensen (1975), commercial strains that have been utilized to prepare food, in the United States, have been tested and have not been found to produce aflatoxins. However, where these products are prepared in a household or village industry, and the fungus is just carried from one batch to the next, wild strains capable of producing aflatoxin may contaminate them. In an investigation, in the Philippines, not a single sample was found that was free of aflatoxin. In such communities probably everyone was suffering to some extent from chronic aflatoxin poisoning.

Other Mycotoxins and Other Fungi

The following mycotoxins and their descriptions were also summarized by Christensen (1975), Hunter (1989), Hudler (1998) and Kendrick (1992). 

Aspergillus ochraceus and ochratoxin

Aspergillus ochraceus is also a species complex consisting of many species. Production of the mycotoxin ochratoxin, by A. ochraceus, was first described in South Africa by Theron, et al. (1966), where it was isolated along with a number of other fungi. In experiments done with this isolate, the LD50 (the single dose that will kill 50 percent of the individual animals tested) of ochratoxin for rats is 22mg/kg (= 22 milligrams of the toxin per kilogram of body weight of the rat), but a lesser amount will result in severe liver damage. A single dose of 12.5 mg/kg (=12.5 milligrams of the toxin per kilogram of body weight of the rat) was administered to pregnant rats on the tenth day of gestation, and of the 88 fetuses involved, 72, or 81.8% died or were dissolved. Ducklings seem to be equally sensitive to ochratoxin as they are to aflatoxin.

Another fungus, Penicillium viridicatum, can also produce ochratoxin, and is relatively common in stored corn and is a more common producer of ochratoxin than A. ochraceus.

Aspergillus fumigatus and fumagillin

This particular species is also known to be an animal pathogen. Infection occurs through inhalation of spores and affects the lungs. Infection may also occur in eggs and the fetuses of cows.  However, it also produces a mycotoxin. This species differs from the others that we have discussed in that it is said to be thermophilic, that is, it is found in substrate where there are extremely high temperatures, up to 122ºF (=50ºC). This species is usually found on material that is in the advanced stages of decomposition in which the substrate temperature has been significantly raised by microbial decomposition.

Under the proper conditions, A. fumigatus produces fumagillin. This compound was originally used as a treatment against Nosema apis, a microsporidial fungal pathogen of honey bees and has proven itself effective against human amoeba parasites as well as microsporidial diseases (Wikipedia 2011). However, it is also a mycotoxin and can inhibit neutrophil function, thereby inhibiting the immune system. 

The Genus Fusarium

Species of Fusarium are widespread in nature as saprotrophs in decaying vegetation and as parasites on all parts of plants. Many cause diseases of economically important plants. For this reason, there has been a great deal of research carried out in this genus by both plant pathologist and mycologist. However, there are a number of species that produce mycotoxins, with trichothecenes being the most studied. 

Fusarium tricinctum

The effects of the first trichothecene toxin, T-2, was documented in the 1940s where it was associated with an outbreak of alimentary toxic aleukia (ATA). At its peak, in 1944, the population in the Orenbury District and other districts of the then USSR suffered enormous casualties, more than 10 percent of the population was affected and many fatalities occurred. The term alimentary toxic refers to the toxin being consumed in foods and aleukia refers to the reduced number of white blood cells in the affected person. Other symptoms included bleeding from nose and throat, multiple, subcutaneous hemorrhages.

The infected food in this case was millet, which made up a great part of the diet of the people in the region, and at times, during WWII, it was not uncommon to allow the millet to be left standing in the fields over winter because bad weather in the fall prevented its harvest at the proper time. During the late winter and early spring the millet would become infected with a variety of fungi, including F. tricinctum, and when the people gathered and ate this fungus, many came down with what was diagnosed as ATA. Thousands were affected, and many died. Locally, Joffe, a plant pathologist determined the outbreak of ATA was caused by consumption of a toxin, present in the millet, which had been contaminated by F. tricinctum. This was a remarkable conclusion since this was 20 years before aflatoxin was discovered. However, Joffe did not isolate or identify the toxin involved and as a result his work remained unknown until 1965 when he presented a summary of his research at a symposium on mycotoxins. The mycotoxin involved was later given the common name T-2, and classified as one of several trichothecenes. Fed orally to rats, it has an LD50 of 3.8mg/kg, which is lower than that of aflatoxin, but still quite toxic enough. LD50 is the term given for a toxicological tests where toxins are administered to laboratory animals to obtain the degree of toxicity. The LD50 represents the dosage of a toxin that will kill 50 percent of a population of laboratory animals. Because this is a standard unit that measures toxicity, it can be used to compare relative toxicity of different mycotoxins.

Fusarium graminearum

Corn is a stable in many countries and is used as a major ingredient in preparation of food for pigs and other domestic animals. Like many other grains, the kernels can be infected with fungi before and after harvest, and can affect the nutritional value of corn as food or feed.

If the weather is rainy and the ears of corn are maturing in late summer and early fall, F. graminearum may infect only a few to a third of the kernels. Whatever amount of the ear is infected, all the kernels in that portion becomes heavily infected and decayed by the fungus. This fungus-infected corn is unattractive to pigs, as well as other animals, and they refuse to it. For this reason, this phenomenon has been called a refusal factor. Regardless of what the composition of the rest of the feed, if it contains more than 5 percent of kernels with this refusal factor, the pigs will not eat it and weight loss will occur. They will starve rather than consume it. The infected corn contains an emetic compound produced by the fungus, and if this corn is consumed by pigs, they suffer prolonged vomiting, after which they sensibly refuse to eat more of the corn (who said pigs were stupid?). The toxin involved is deoxynivalenol (DON), also known as vomitoxin. The isolation and identification of this toxin has occurred only within the last 25 years.

This is a serious problem if you look at it through the eyes of the farmer. If you are a farmer and you have 800-1000 pigs and several tons of feed mixed with corn, contaminated with vomitoxin, was delivered to the farmer's feed bin on a Friday, and it is later determined that the pigs will not eat it, then the farmer has a serious problem. What are the pigs going to eat between Friday and Monday?

Various methods have been tried to make the vomitoxin contaminated corn more acceptable to pigs. Among some of the means that have been tried are adding molasses to the feed to conceal whatever flavor or odor makes it unacceptable to the pigs, heating the feed, in hopes of destroying or inactivating whatever it is that is making the pig refuse to eat it, and composting it so that the heat will break down the toxin. However, none of these treatments have made the corn acceptable to pigs and are impractical, anyway.

The detection of infected corn or feed is also a problem. Since we are talking about mycotoxin here, the inability to isolate the causal agent, F. graminearum, is not evidence that the mycotoxin is absent. Long after a fungus has died off, mycotoxin secreted into the substrate, will still be present. The refusal of pigs to eat feed or corn is an indication that the refusal factor is present, but not necessarily conclusive. There are a number of reasons as to why pigs will refuse to eat. Pigs may be traumatized by being moved to a new pen, strange surroundings or even being offered different food. The only way that the toxin can be detected is to isolate, purify and identify it by spectrographic or other analysis.

Use of Trichothecenes in Biological Warfare

Yellow Rain

For those of you who enjoy conspiracy theories, yellow rain is a good one. During the mid 1970s, when Vietnam was invading Laos, there were stories of "yellow rain" in areas where Hmong villages were attacked. One eye witness account of such an event was told by a Hmong refugee, in Thailand. While tending his poppies, outside of his village, he and his family witnessed the bombing of their village by the Vietnamese, in MIGS, with a yellow powder that came down like yellow rain. Returning to the village, he found all of the animals and most of the people were dead. The bodies were bleeding from the nose and ears and their skin were blistered and yellowed. The few people left alive, when he arrived, were "jerking like fish when you take them out of the water". These people also eventually died. The witness took his family away from the village, but as they left they felt shortness of breath and sick to their stomach. This story is similar to other stories that were heard concerning yellow rain among Hmong refugees. The animosity between Vietnam and the Hmong was due to the latter supporting the United States military during the Vietnam War. 

It was believed by the United States at that time that the Soviet Union was somehow involved in the attacks of the Hmong villages, and medical teams were sent to investigate. However, because of the remoteness of these villages, news of such attacks normally took 4 to 6 weeks to reach someone who could notify the medical teams. By the time investigators reached a village, there was no evidence as to what happened. It would not be until 1980 that a Defense Department chemist diagnosed the symptoms described by victims of the bombing as similar to trichothecene mycotoxicoses. Samples from victims and from vegetation in the areas were tested and some were found to contain trichothecenes, specifically the T-2 trichothecene. With this information, in 1981, Secretary of State, Alexander Haig accused the Soviet Union of violating the Geneva Convention of 1925 and Biological Weapons Convention of 1972, which of course they denied. According to Haig, in the late 1970's, Vietnam and Laos mounted attacks on Hmong Tribes People of Northern Laos with a toxic agent dropped from low flying aircrafts. Eyewitnesses described this agent as an oily yellow liquid making sounds like rain when it fell that stuck to the leaves of trees . Those exposed to liquid were reported to have had heavy bleeding from the nose and gums, blindness, tremors, seizures and other neurological symptoms. Death from exposure also occurred.  

These accusations would continue to be very vocal for three more years. While the accusations and denials were aired, the media and scientific community gave a more critical examination of the yellow rain story and the credibility of these attacks soon became questionable.  The analysis that demonstrated trichothecenes were being used was initially based only on a single leaf, collected where one of the chemical attacks occurred. Subsequent specimens were collected later that also showed trichothecenes were present, but the concentration of trichothecenes differed where it was found and was entirely absent in some samples. In fact, over 100 samples analyzed by the United States Army, which did not find any traces of T-2. While victims examined were found to be suffering from T-2 poisoning, these poisoning were said to be the result of ingestion of the toxin rather than absorption through the skin or inhalation. Eyewitness accounts also came into question. Although it was implied that many villages were attacked with yellow rain, most of the witnesses were from a single refugee camp in Thailand, and their accounts were inconsistent and thought to be unreliable. For example in relating a story of the bombing, one villager had initially said that 213 villagers were killed, but in a later retelling, there were only 13 people killed and then 40. Even the researchers from the United States were questioned as to their investigative methods. It was learned that before interviewing the potential witnesses to these bombings were questioned it was already well known to them as to the reason as to why they were to be questioned. When questions, witnesses were asked leading questions, e.g. "Describe the poisonous attack", witnesses were not selected at random, i.e. only those villagers who said they witnessed attacks were interviewed, investigators did not take samples, but instead allowed villager to gather them, lack of control groups, etc. Another words, the researchers violated proper forensic methods while investigating the cause of yellow rain.

Further erosion of the government's yellow rain story came about when a scientist from the British Chemical and Biological Defense Establishment, at Porton Down, England examined actual samples of yellow rain specimens, under the microscope and determined that most of the it was composed of pollen grains. The collection was made in 1979, but microscopic examination did not occur until 1982.This observation would later be made independently by scientists in Australia, Thailand, Canada, France and Sweden. Eventually this observation would be acknowledged by scientists at the U.S. Army's Chemical Systems Laboratory (CSL). The announcement as to the composition of yellow rain was made by Emory Sarver of the CSL who described samples as being composed of the toxin, organic solvents and pollen. A detailed explanation of the yellow rain composition was made later by Sharon Watson, an intelligence analyst for the government. She explained that the yellow rain had two modes of transmission to the victims. One was by absorption by skin through direct contact with the solvents that contain the T-2 and the other was by inhalation of pollen grains that would remain afloat in the air where they would be inhaled and taken to the victim's lungs.

After it was determined that yellow rain was composed of pollen, Joan Nowicke, a palynologist at the Smithsonian examined the pollen grains and determined that they originated from plants that were local and commonly found in the areas where the attacks were reported to have taken place. This would seem unlikely if the yellow rain was produced in the Soviet Union. By this time it appeared more and more likely that yellow rain was a natural phenomenon rather than a biological weapon.

Yale University entomologist, Thomas Seeley, whose expertise was in Southeast Asian bees, would provide the final piece to the puzzle. In 1984, Seeley led a group on a field study in Thailand where they observed the swarming of a larger number of wild honey bees that flew high into the air until almost invisible and en masse defecated for several minutes, covering an acre or more with hundreds of thousands of yellow spots onto the landscape that were identical in appearance to yellow rain. Such mass defecations were seen at several localities and when the research group showed samples to Hmong refugees, who said they had witnessed yellow rain attacks, they identified the fecal droppings as what they were calling yellow rain.

In summary, there may not have been a conspiracy. Instead, it seems likely that in investigating attacks of the Hmong, by Vietnam and Laos, was carried out in an unscientific matter, where an unknown phenomenon, i..e. yellow rain that was witnessed was thought to a biological weapon and no other avenue was explored. While the number of deaths during these attacks could not be disputed, the utilization of conventional weapons, such as explosives, phosphorous and napalm, to name a few could be the reasons for these deaths. 

However, While a plausible alternative was given as to the cause of the yellow rain, representatives of the United States responded to most of these contradictions in their findings. Most of the arguments are summarized in Wannemacher and Wiener (1997). The latter authors disputed that the eyewitness accounts were inconsistent, that descriptions of the symptoms were consistent with exposure to trichothecenes, while pollen was what was found on leaves that trichothecenes were present on the pollen and 32% of samples tested from victims tested positive for trichothecenes, far too high for it to be due to ingestion. In addition, they recounted confessions by a defecting Laotian Air Force officer and North Vietnamese ground troops that they had used chemical weapons and their description of how the weapons were used were consistent with descriptions by eyewitnesses. However, documentation of the confessions was not given. One piece of evidence that has been frequently used to support the use of trichothecenes is that while the bees are still defecating en masse, no further deaths have been reported. 

To date, the United States continues to maintain that biological weapons were used during the yellow rain incident. What actually occurred during the years in which yellow rain occurred may never be known. However, President Reagan used the incident to the advantage of the defense budget. Despite denial of biological warfare by the Soviet Union, President Reagan continued with his accusations for three years and with the atrocities that were being reportedly caused by trichothecene weapons in the news media, he was able to solicit large increases in the defense budget to develop defensive chemicals against biological weapons. 

Trichothecenes and The Black Death

In order to understand the disease, let us first go over its life history. The bacterium, Yersinia pestis, is the actual pathogenic agent that causes the The Black Death (=Bubonic Plague). However, it does not directly infect humans, most commonly, Xenopsylla cheopis, a species of flea that specifically infects rats is the carrier of the disease. Pulex irritans, a flea that typically infects human can carry also carry the disease, but this is uncommon. The disease cycle begins when the bacterium enters the stomach of a flea that has bitten an infected rat and dined on its blood. If the rat host dies of the disease or for some other reason, the flea will have to find another host. If the flea should bite a human and sucks its blood, it regurgitates blood and plague bacilli into the bite site thereby infecting its human host.

Symptoms of Disease

The Black Death can be readily be recognized by the large, swollen lymph nodes (=Buboes). These may occur on the neck, arm pits and groin. Other symptoms include headaches, chills, fever, nausea, vomiting, aches in legs and back.

Figures 1-2. 1: Population of Europe following Black Death, from http://www.schillerinstitute.org/graphics/graphs/gallagher_population_article/fig-6.jpg. 2: Fresco depicting victims of the Black Plague, from http://en.wikipedia.org/wiki/File:Plague_-buboes.jpg.

 

The Plague

It was believed that during the High Middle Ages, the 1100s-1200s, Europe was in a period of relatively good health and population growth. However, this growth ended between 1348-1350, when a major epidemic of the Bubonic Plague struck. It is estimated that 1/3 of Europe’s population died as a result of the plague. While Matossian (1989) did believe that the deaths to be attributed to The Bubonic Plague, she also believed that because of the extraordinary, large number of people that died during this period that the disease organism would have to be either extremely virulent or that the immune system of the human host had been compromised by consumption of grain contaminated with T-2 and other mycotoxins. Her conclusion was based symptoms that were consistent T-2 ingestion rather than the plague. Other symptoms also indicated other diseases may have been involved (Dees 2008).  

Matossian also believed that there was an increase in death of rats, from consumption of contaminated grains. With greater number of rats dying, more fleas would require new hosts, and in heavily populated area, often this would be a human host. This would lead to a higher number of people contracting the disease than might have normally occurred. She also presented evidence, based on what seemed to be selectivity of the disease, based on age and wealth, grain storage and environmental moisture.

Due to the cold and wet years that occurred in 1348-50, in certain areas of Europe, grain crops, which were the staple for Europe at this time, were thought to have been contaminated with T-2 or related toxins that damaged the immune systems of both rats and humans. The damage to the immune systems of both rats and human is is believed to be one the contributing factors that led to the high mortality during the Bubonic Plague. However, other causes of depressed immune systems, other than fungal in origin, may also have occurred at this time.

Most of this lecture was based on the following books. If you are interested in reading a more detailed account of the above stories, you may find them in Hamilton Library.

Christensen, C.M. 1975. Molds, Mushrooms, and Mycotoxins. University of Minnesota Press, Minneapolis. 264 pp.

Dees, R. P. 2008 Economics and Politics of Peasant Production in South Germany, 1450--1650. UMI Dissertation Service.

Elshafie, S.Z.B., A. ElMubarak, S.A.F. El-Nagerabi and A.E. Elshafie. 2011. Aflatoxin B1 Contamination of Traditionally Processed Peanuts Butter for Human Consumption in Sudan. Mycopathologia 171:435-439.

Hudler, G. 1998. Magical and Mischievous Molds. Princeton University Press. 248 pp.

Hedayati, M.T., A.C. Pasqualotto, P.A. Warn, P. Bowyer and D.W. Denning. 2007. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 153: 1677-1692.

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Mycological Terms:

Mycotoxins: In broad sense, toxic substance of fungal origin. As used in this lecture, fungal metabolites that are toxic to humans and/or animals that are produced by molds growing on foodstuffs.

Molds: Fungi usually referable to Fungi classified in the Form Phylum: Deuteromycota, a phylum erected to accommodate those fungi that have septate mycelium where sexual reproduction is unknown or rare.

Conidia: Asexual spore that is not produced in a sporangium and borne on modified, upright hyphae called conidiophores.

Conidiophores: Modified, upright hyphae on which conidia are borne.

Deuteromycota: A phylum that has been created to classify those Fungi that form septate mycelium, but are not known to have sexual reproduction and are known to only reproduce asexually by conidia.

 Aflatoxin: First mycotoxin discovered and the cause of what was called the Turkey -X disease that was initially thought to be caused by a virus.

Turkey -X disease: What was initially thought to a viral disease that killed over 100,000 turkeys and a number of other domesticated birds, but later discovered to be poisoning of contaminated peanut, in food, due to aflatoxin poisoning.

Aspergillus flavus: Mold from which aflatoxin was discovered and named, i.e. Aspergillus flavus + toxin = aflatoxin.

Trichothecene -2 (T-2): Mycotoxin that caused the death of 10% of population of Russia during and after World War II, in the 1940s. Death was thought to be due to alimentary toxic aleukia. Actual cause of deaths not known until 1960.

Yellow Rain: A name given to what was believed to be T-2 mycotoxin dropped from low flying planes onto Hmong villages by Vietnam and Laos. The name was believed to have originated because the liquid dropped from the planes was yellow and when it hit the ground, made a sound like that of rain drops.

Questions to Think About