Mycotoxinas en Cereales

8/10/2019 Mycotoxinas en Cereales

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Grain Fungal Diseases& Mycotoxin Reference


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Preface

This manuscript is a compilation of information that has been available to college professors,researchers, and grain trade specialists for some time. We will try to present this information in away that will be helpful to non-specialists in an easy to digest manner. The nature of this informa-tion tends towards the technical and sometimes keeps needed information out of the reach of thosethat could use it most. We will try to keep this in mind while presenting the needed information.Also, while not being technically correct, we have kept reference citations to a minimum, and haveinserted numbers, i.e.: [ 23], to keep the interruptions to the flow ideas as few as possible.


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Grain Inspection, Packers and Stockyards AdministrationTechnical Services Division

Mission

TSD provides technical leadership, training, and support services;inspection methods development; specialized analytical tests;

national quality assurance and standardization processes;and final appeals

for the benefit of GIPSAs grain inspection customers.

Vision

The Global Leader in Grain Quality Assessment .

GIPSA, Technical Services Division10383 N. Executive Hills Blvd.

Kansas City, MO 64153(816) 891-0401

FAX (816) 891-8070


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Contents

Chapter 1: Fungi and Fungal Diseases of Plants ........................................................5

Chapter 2: What Are Mycotoxins? .......................................................................8

Chapter 3: Mycotoxins in Grain and Feed ...............................................................14

Chapter 4: Sampling ...............................................................................................24

Chapter 5: Analytical Procedures ............................................................................30

GIPSA Approved Mycotoxin Methods ...................................................................36

Glossary ...................................................................................................................37

References ...............................................................................................................40

Internet Resources ...................................................................................................42

Fungal Disease Fact SheetsAflatoxin ........................................................................................................46Black Tip .......................................................................................................47Blue-Eye ........................................................................................................48Ergot ................................................................................................................49Fusarium Ear Rot .......................................................................................50Gibberella Ear Rot .....................................................................................51Karnal Bunt ...................................................................................................52Scab ...........................................................................................................53TCK Smut .................................................................................................54


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Chapter 1

Fungi and Fungal Diseases of Plants

Definition and Overview. Fungi are prob-ably one of the most numerous plant familieson earth. By definition they are plants thatcontain no chlorophyll (can grow in condi-tions of little or no natural light) and rangefrom single cells to a body of branched hy-phae (tubular filaments) that often producefruiting bodies that form molds, mushrooms,smuts and yeasts. Instead of producing theirown food, fungi absorb nutrients from eithera living or dead host material. Symptoms anddisease development come about from thegrowth of the fungi through the host-parasiteinteraction. These fungi sometimes producemetabolites (by-products of growth) that aretoxic to animals and humans. Reproductionin fungi occurs through the production of spores. These spores can then reproduce with-out coming into contact with a different plant(asexual reproduction).

The small size of the spores aid in their dis-persal. They can become airborne and moveby the action of winds and travel from field tofield. They also can become attached to in-sects and birds which then transport them fromplant to plant. Transport can also occur byuse of contaminated trucks and equipment.Fungal infection from spores can occur at anyof the various stages of crop production. Itcan begin in the fields, in or on the crop itself.It can infect healthy products during transpor-tation and storage by coming into contact withcontaminated equipment or grain products.The spores can lay dormant (inactive) in thesoil or accumulate on equipment or in storage

facilities for months or sometimes years untilthe proper conditions for growth occur andinfect generation after generation.

There are many varied environmental condi-tions that need to be in place before the sporeswill germinate or begin to grow. Generally rela-tive humidity over 70% and temperatures over30 C (86 F) for extended periods (severaldays to a week) are generally needed. Stressto the plants such as periods of drought, flood-ing, or insect infestation are also common fac-tors in the fungus growth cycle. High mois-ture content of the crops (20% or higher incorn), as at the optimal times of growth andharvest, give the spores the necessary elementsto start the growth process.

Any one of these conditions by itself will notpromote the fungal growth. It is only when

the right conditions as a group occur, for eachparticular fungus, do the growth cycles begin.If any one optimal condition is removed, thegrowth cycle of a particular fungus will stop.But this may then promote the growth of adifferent or related fungus. Once the growthcycle begins the crop damage has alreadystarted and can not be reversed. The dryingof corn lowers the moisture content to a pointwhere growth of molds is prevented or at leaststops further damage.

Fungi and mold are normally thought of asmushrooms and the grey fuzzy stuff that growson food left in the refrigerator too long. Moldsand fungi do not necessarily have to be


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visible to the naked eye to be growing anddamaging crops and foods. Many of the sporesassociated with fungi are microscopic and intheir first stages of growth are not visible with-out the aid of a microscope. Only in the later

stages or when large masses of fungi arepresent, do the fungi become visible to theunaided eye.

The presence of visual mold in grain or feedproducts does not necessarily mean that myc-otoxins are present in the sample. Moldgrowth indicates that there are some fungipresent. Many of the fungi grow under simi-lar conditions and more than one kind of fungican exist in or on the grain or feed product atthe same time. When more than one fungi ispresent at the same time on the same host, theysometimes increase the toxic effects of eachother by attacking different bodily functionswhen ingested by a human or animal. Theycan also cancel each other out by growing andattacking each other during the growth pro-cess. The lack of visual mold does not meanthat there are no mycotoxins present. Manytimes during harvest or handling the visual

aspects of the mold can be brushed off, butthe toxic by-products can remain in the ker-nel.

Non-Mycotoxic Fungi

Fungi are a major cause of spoilage in storedgrain. The Food and Agriculture Associationestimates that 25% of the worlds food cropsare affected by mycotoxins (the by-productsof fungal growth) during growth and storage.The damage of fungi is second only to thatcaused by insects in stored grain products.Many of the fungi cause damage to the cropsthemselves with little or no toxic effects onhumans and animals [ 15 ].

Common Smut or Bunt. Common smut iscaused by two fungi; Tilletia tritici and Tille-tia laevis. Because it requires cool moist soilconditions, the disease is less of a problem onspring planted wheat than winter wheat. Com-

mon smut reduces wheat yields and grain qual-ity. Wheat contaminated with bunt sporeshas a pungent, fishy odor and a darkened ap-pearance. Wheat that has an unmistakableodor of smut will be designated light smuttyon official inspection certificates. In addition,bunt spores released during combining arecombustible, and have caused explosions andfires during harvesting with mechanical har-vesters. [23]

Plants may be moderately stunted but are noteasily distinguished until the heads emerge.Bunted heads are slender and maintain theirgreen color longer than healthy heads. Theglumes may spread apart exposing the smutballs they contain. Smut balls are approxi-mately the shape of normal kernels and aredull gray-brown. The small balls often rup-ture at harvest, releasing black, powderyspores. Wheat containing smut balls will be

designated as light smutty (6-30 smut balls)or smutty (31 or more smut balls) on offi-cial inspection certificates.

Dwarf Bunt or TCK Smut . The Tilletia spe-cies of fungi cause smut or bunt diseases inwheat, rye, and barley. Tilletia controversa ,commonly known as Dwarf Bunt or TCKSmut, causes dwarfing or stunting in thegrowth of the plant itself along with reducedyields. The major source of transmittal is by

the spores laying dormant in the soil until thenext crop year. These spores can lay dormantfor up to ten years retaining their infectiousproperties [ 23].


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Karnal Bunt . Karnal Bunt is thought to haveoriginated in India (therefore the name Tille-tia indica ) but has since been discovered inmany places worldwide. It is thought to be inAfghanistan, Lebanon, Mexico and now the

United States. Karnal Bunt has spores thatare primarily wind-borne, but infection canoccur through planting in contaminated soil,use of infected seed, and coming into contactwith contaminated equipment and machinery.The spores attack the developing kernelswithin the seed head with little or no outwardsign of infection. The infected kernels areshrunken at the germ end and are covered withsori (small hairlike filaments) that will discolorflour made from infected kernels. These ker-nels will also impart a fishy odor and taste toflour making their commercial use impracti-cal [10, 23 ].

Black Tip / Black Point . Black Tip fungusis another non-mycotoxic fungus that attackswheat and barley. The name Black Tip orBlack Point can generally refer to any of anumber molds that form dark brown to black sooty mold. The principal species of infec-

tion are Al terni a, Fusari um, and Helmin thosporium , with the species Helminthosporium generally being the causalagent in infections associated with wheat [ 23].The mold generally occurs when seeds aresown in infected soils, but can also occur whenthe seeds themselves are infected. The mostcommon forms of Helminthosporium are gen-erally associated with dry, warm soils. Mois-ture in the form of rain or extended periods of high humidity (over 90% relative humidity)

then start the disease on the maturing kernels.If the mold begins early in kernel growth ste-rility and germ death occur, often accompa-nied with a shriveled germ resulting in de-creased yields. If the mold attacks in the later

stages of kernel development the seed is dis-colored and often carries an odor causingmarket discounts.These molds are also associated with seedlingblight and root rot which will effect the over-

all health of the plant leading to increased sus-ceptibility of other plant diseases and molds.

Blue-Eye Rot. Blue-eye mold occurs instored corn with high moisture content. Blue-eye damage is caused by species of Penicil-lium and is characterized by a blue-green dis-coloration in the germ area. The discolora-tion results when Penicillium fungi invade thegerm area through the tip of the kernel. Somecorn varieties have a purple colored plumulewhich can be mistaken for the presence of Penicillium fungi in the germ area.

Corn Smut. Smut is caused by Ustilagemaydis and is always present in field corn. Noharmful effects have been noted from feedingsilage made from smutty corn to livestock.


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Background. In the previous chapter the prin-cipal damage caused by the fungi was themold. There were little or no toxic affects fromby-products of the mold. The molds were alsolimited as to the hosts they attacked. Mycotox-ins on the other hand are metabolites (by-prod-ucts) of the growth of the molds. They havevery real toxic side effects to other plants, ani-mals, and humans. They are also generallyless selective of the hosts they attack and cancross plant species.

The species Fusarium can and will attack bothcorn and wheat with different effects in eachplant. In wheat, they cause scab damage tothe kernels and produce deoxynivalenol(DON). In corn, they create Gibberella EarRot and produce DON, zearalenone (ZEN) andT-2 toxins. Both DON and ZEN have toxiceffects on animals and humans, with differ-

ences depending on the species.

Mycotoxin contamination of crops has beena world wide problem for thousands of years.Only in the last thirty or forty years has tech-nology allowed researchers to isolate the fun-gal mycotoxins and study the effects on feedcrops, livestock, and their affects on humans.The study of mycotoxins is a narrow part of the research into naturally occurring toxins andtheir effects on plants, animals, and humans.Much of the information in print becomesquickly dated as the field expands and grows.With recent improvements in testing methodsmore research is now being done, and ingreater detail, with lower costs than was pre-

viously possible. Many of the newest researchpapers are now posted on the Internet allow-ing new information to reach more peoplefaster and more efficiently than was possiblebefore. Information that was published only20 to 25 years ago is now considered datedand questionable. But with the limited natureof the end usefulness of the information allinformation must be considered.

The problems associated with mycotoxin con-tamination of grains are world wide and areuncontained by national borders. A look at amap of North America shows that the impor-tant grain producing areas stretch from northcentral Canada to the southern reaches of theUnited States. These areas in turn export theirproducts to countries in Asia, the Pacific is-lands, South and Central America, Europe, theMiddle East, India and Africa [ 15 ].

Scientists estimate that there are 300 to 400mycotoxins presently identified with morebeing isolated as new techniques and processesevolve. The most frequently found mycotox-ins are aflatoxin, deoxynivalenol (DON),zearalenone (ZEN), fumonisin, and T-2 as faras grain crops are concerned. In surveys con-ducted by the North Carolina CooperativeExtension service in 1990 some amount of aflatoxin, DON, or fumonisin was found inover 70% of the samples tested [ 20 ].

Mycotoxic molds generally attack the kernelsof grain robbing the nutrients and lower thefat, protein, and vitamin content of the grain.

Chapter 2

What Are Mycotoxins?


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The mold also often changes the color of thekernels, the consistency (texture), and oftenimparts an odor that causes feed refusal inanimals. These effects lead to economic lossesdue to impaired health in animals and humans,

reduced productivity (reduced production of eggs, milk, and weight gain), and in severecases death to animals and humans. Whenthe grain is processed into final products likeflour or feed, the visible mold may be removed,but the majority of toxins are not and can stillcause poisoning.

Economic effects. The economic effects at-tributed to mycotoxin infection are widely feltin all sectors of the production and consump-tion of grain products. Grain producers areaffected by limited yields, restricted end mar-kets, and price discounts. Grain handlers areaffected by restricted storage options, cost of testing grain lots, and loss of end markets.Grain processors incur higher costs to due tohigher product losses, monitoring costs, andrestricted end markets. Consumers end uppaying higher end product prices due to in-creased monitoring at all levels of handling,

and in extreme cases health problems due toconsumption of contaminated products. So-cieties as a whole end up paying higher costsdue to increased regulations, needed research,lower export costs, and higher import costs.

While these costs are found at every level of the grain production system, it is almost im-possible to put a dollar figure on the losses.Estimates of losses to small portions of thegrain and related industries are the best that

can be accomplished. The North CarolinaCooperative Extension Service published anestimate for 1992 that the losses to the animalproduction industry in North Carolina were$20 million for poultry, $10 million for swine,

$5 million for dairy, $1 million for beef andsheep, and $1 million for horses. In 1990 avomitoxin (DON) outbreak in New York andother Northeastern states, the NorthcentralU.S., and Eastern Canada had widespread eco-

nomic affect. The New York Corn Growersestimated, conservatively, that $12 milliondollars was lost by the corn farmers due tolost markets, decreased crop value, and costsassociated with testing for the 1990-91 cropyear [15,20 ].

Health Hazards. The health hazards associ-ated with mycotoxin contamination in humansare rarely seen in North America and Europe.This is generally attributed to a higher levelof general health than is seen in underdevel-oped countries and better control of food andfeed storage.

The levels of intake of affected products thatare necessary to bring about poisoning inhealthy individuals are actually quite high. Inthe rare cases that humans have been reportedto be poisoned by mycotoxins, the populationswere consuming limited quantities of other

non-tainted foods and lived in areas of eco-nomic and environmental stress. The great-est threat of health hazards to humans comesfrom long term exposures of tainted food prod-ucts, either from spoilage or from consumingmilk or meat from animals that have been fedcontaminated feed [ 6, 12 ].

One possible avenue of concern to humans isthe suspected link between aflatoxin and can-cer. While the International Agency for Re-

search on Cancer (IARC) has listed aflatoxinB1 as having a definite link to cancer in ani-mals, it is listed as having a probable link tocancer in humans. The studies that have beendone on the cancer link to humans have been


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done in Africa and Asia and show anassociation between aflatoxin and cancer butno definitive cause and effect relationship hasyet been documented [ 21 ].

Mycotoxicoses (poisoning due to mycotoxins)have several common symptoms that areshared from species to species and toxin totoxin. These symptoms include:

1. Drugs and antibiotics are not effective intreatment.

2. The symptoms can be traced (associated)to food or feedstuffs.

3. Testing of food/feedstuffs reveals fungalactivity. [ 12 ]

4. The symptoms are not transmissible tocontrol subjects.

5. The degree of toxicity in subjects isinfluenced by age, sex, and the nutritionalstatus of the host.

6. Outbreak of symptoms is seasonal.

Mycotoxins have been linked to birth defectsin many animals, nervous system problems(tremors, limb weakness, staggering, and sei-

zures), and tumors of the liver, kidneys, uri-nary tract, digestive tract, and the lungs [ 6 ].

Aflatoxin primarily attacks the liver, with sec-ondary effects shown in decreased production,and immune system suppression. Thetrichothecene group of mycotoxins (DON andT-2) cause necrosis and hemorrhage of the di-gestive tract with decreased blood productionin the bone marrow and spleen along withchanges to the reproductive system as the sec-

ondary sites of attack.

Zearalenone has affects different from othermycotoxins in that it mimics the bodies pro-duction of estrogen causing feminization of

male animals and interference with concep-tion, ovulation, and fetal development infemale animals [ 6 ].

One of the primary similarities in mycotoxi-

cosis is the health status of the host beforeinfection, which will to a large extent deter-mine the degree to which the host is attacked.In both animals and humans if the host ishealthy the final prognosis is generally verygood except in cases of acute poisoning wherevery large doses of toxin are eaten over a veryshort period of time.

Many variables affect the degree of suscepti-bility of various hosts. These include:

General health . A healthy individual is moreable to fight the toxins than a one that startsout malnourished or diseased.

Age . The very young and very old have weak-ened immune systems that are less able to fightthe effects of toxicoses.

Sex . In general female animals, and to a de-

gree female humans, seem to be more suscep-tible to the effects of mycotoxicoses.

Environment . Hosts exposed to harsh livingconditions of neglect or squalor have an addedburden on their systems.

Adequate food storage . If grain is left in openstorage to the effects of weather the grain isfurther weakened allowing for continued fun-gal growth. If the grain is also not adequately

dried, the conditions for fungal growth con-tinue to persist.

Exposure Level . Very high doses of aflatoxinattack the host quicker than lower doses.


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Exposure duration . A very short time of ex-posure (a single dose) allows the host time tofight the infection where a continued expo-sure tends to have cumulative effects.

Other food sources . If contaminated grainis the only or main source of nourishment, thehost has less nutrients available to feed theirsystem.

Lack of Regulatory and Monitoring sys-tems . Areas that lack the means to regulateand monitor the presence of mycotoxins ingeneral, and aflatoxin in particular, leave theirinhabitants open to unknown poisonings thatcan continue over long periods of time un-checked. [21]

Although the mycotoxins have been greatlyresearched over the course of the last fortyyears, little research has been done on the in-teractions of the mycotoxins and their com-bined effects. Almost all research has been indetermining the effects of pure strains devel-oped in the laboratory. Animal feed can con-tain several different grains and grain in stor-

age can contain several different strains of thevarious mycotoxins.

Studies done with natural field contaminatedDON and ZEN have produced results thathave varied from those that have been carriedout in the laboratory. This leads researchersto believe that there are unknown strains of toxins in the field or that the toxins interactwith each other to produce effects greater thanor different from what laboratory tests have

predicted would occur [ 15].

Regulatory Control . In 1965, the Food andDrug Administration (FDA) established actionlimits of 20 parts per billion (ppb) of aflatoxin

in all food and feeds to limit the inclusion of this contaminate into the food chain. But sinceaflatoxin can occur both in raw products andin finished by-products, this has necessitatedthe testing for aflatoxin and other mycotoxins

at many points in the food chain. As technol-ogy has progressed in recent years testing hasbecome simplified, faster, and cheaper (us-ing ELISA methods) allowing more testing tobe accomplished in all phases of food han-dling.

The Grain Inspection; Packers and StockyardsAdministration (GIPSA) tests all corn that isexported for aflatoxin with an action level of 20 ppb. If a sample tests above 20 ppb, theFDA must be notified. Testing for vomitoxinis done as a service to customers with no FDAaction level set. FGIS will institute new test-ing services as the tests are certified for reli-ability and repeatability, and as customer de-mand requires.

Aspergillus Toxins

Aspergillus is an important genus in foodswith most species occurring as spoilage orbiodeterioration fungi. Aspergillus is a largegenus containing more than 100 recognizedspecies, several of which are capable of pro-ducing mycotoxins.

Nearly 50 species of Aspergillus have beenlisted as producing toxic metabolites. Thoseof greatest significance in feed and foods in-clude: aflatoxin, ochratoxin A, sterigmato-cystin, cyclopiazonic acid, citrinin, patulin,and tremorgenic toxins.

Aspergillus species produce toxins that ex-hibit a wide range of toxicities, with the most


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significant effects being long term. AflatoxinB1 is a potent liver carcinogen. Ochratoxin Aand citrinin both affect kidney function.Cytopiazonic acid has a wide range of effectsand tremorgenic toxins affect the nervous sys-

tem.

While there is a known link between aflatoxinand cancer in animals, it should also be notedthat the Aspergillus species of fungi also havemany beneficial uses. One of the largest com-mercial uses of Aspergillus fungi is in the pro-duction of soft drinks. The extraction of purecitric acid from fruits and vegetables hasproved to be to expensive, so the manufactur-ers have developed a way to use large vats toferment Aspergillus niger to form artificialcitric acid [ 22 ].

The Japanese have developed methods of fer-menting rice with strains of Aspergillus flavus(aflatoxin) to cause the enzymes in the ker-nels to breakdown the carbohydrates intosimple sugars, to produce a sweetened ricedrink. These drinks or Koji, are marketedunder many different brand names [ 7 ].

The family of products that includes miso, soysauce, and sake also use strains of Aspergil-lus oryzae to ferment grain products into us-able food and drink products [ 22].

Penicillium Toxins

Penicillium is a large genus with over 150 spe-cies recognized and at least 50 species of com-mon occurance. The discovery of penicillinin 1929 gave impetus to a search for otherPenicillium metabolites with antibiotic prop-erties. This search led to the recognition of toxic antibiotics or mycotoxins.

Nearly 100 Penicillium species have been re-ported as toxin producers. Of these the fol-lowing nine mycotoxins produced by 17 Peni-cillium species are potentially significant tohuman health: citreoviridin, citrinin,

cyclopiazonic acid, ochratoxin A, patulin,penitrem A, PR toxin, Roquefortine C, andSecalonic acid D.

The toxins produced by Penicillium speciescan be placed in two general groups: those thataffect the liver and kidney function, and thosethat are neurotoxic. The Penicillium toxinsthat affect liver or kidney function are asymp-tomatic or cause generalized debility in hu-mans or animals while the neurotoxins arecharacterized by sustained trembling.

Fusarium Toxins

Fusarium species are the most importantgroup of mycotoxigenic molds other than As-

pergillus and Penicillium . Many Fusariumspecies are plant pathogens and most can be

found in the soil.

Fusarium species are most often encounteredas contaminants of cereal grains, oilseeds, andbeans. Corn, wheat, barley and products madefrom these grains are most commonly con-taminated although rye, triticale, millet, andoats can also be contaminated.

F. graminearum is a plant pathogen foundworldwide in the soil and is the most widely

distributed toxigenic Fusarium species. Itcauses various diseases of cereal grains suchas gibberella ear rot in corn and head blight orscab in wheat and barley. The mycotoxinsproduced by F. graminearum include:


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deoxynivalenol, zearalenone, 3-acetyldeoxy-nivalenol, 15-acetyldeoxynivalenol,diacetyldeoxynivalenol, nivalenol, T-2,neosolaniol, and diacetoxyscirpenol.

F. moniliforme is a soilborne plant pathogenthat is found in corn growing in all regions of the world. It is the most prevalent mold asso-ciated with corn. It has also been found inrice, sorghum, yams, hazelnuts, pecans, andcheeses.

F. moniliforme has long been suspected of being involved in animal and human diseases.Animal diseases associated withF. moniliforme include equine leukoencephal-omalacia (ELEM) a liquefactive necrosis of the brain of horses, pulmonary edema andhydrothorax in swine, liver cancer in rats, andabnormal bone development in chicks andpigs.

The main human disease associated with F.moniliforme is esophageal cancer. Severalstudies have linked the presence of F.moniliforme and fumonisins in corn to high

incidences of esophageal cancer in humans incertain regions of the world including an areaaround Charleston S.C. Mycotoxins that havebeen associated with F. moniliforme includefumonisins, fusaric acid, fusarins, andfusariocins.

Alternaria Toxins

Alternaria species may be significant as po-tential contaminants of food. Alternaria in-fects the plant in the field and may contami-nate wheat, sorghum, and barley. Alternariaspecies also infect various fruits and veg-

etables and can cause spoilage of these foodsin refrigerated storage.

Al te rnar ia toxins include: alternariol,alternariol monomethyl ether, altenuene,

tenuazonic acid, and the alertoxins. Little isknown about the toxicity of these toxins; how-ever, cultures of Alternaria that have beengrown on corn or rice and fed to rats, chicks,turkey poults, and ducklings have been shownto be quite toxic.

Claviceps Toxins

The ergot mold, Claviceps purpurea , is thecause of the earliest recognized human myc-otoxicosis, ergotism. Ergot has been reportedin sporadic outbreaks in Europe since 857,with near epidemic outbreaks in the MiddleAges.

Ergot is a disease of cereal grains such as ryeand wheat in which the grains are replaced byergot sclerotia that contain toxic alkaloids.The main ergot alkaloid, ergotamine, has vaso-constrictive properties that can cause swollenlimbs, and alternating burning and cold sen-sations in the fingers, hands, and feet (St.Anthonys Fire).


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AflatoxinPathogen. The name aflatoxin comes fromA(Aspergillus) + FLA(flavus) + toxin. Mod-ern research into aflatoxin had its beginningsin 1961, looking into what caused the deathsof 100,000 young turkey poults in England.The research traced the poison to contaminatedBrazilian peanut meal that had been used asfeed. When the feed was given to ducks andpheasants, the same outcome was produced.

Research found that there were four differentmetabolites formed from aflatoxin. When thecontaminated grain in question was viewedunder a black light, the metabolites glowedeither blue (B metabolite) or green-yellow (Gmetabolites). Of the two distinct color variet-ies there were isolated two distinct toxins (B 1,B2, G1, G2). The subscripts refer to their sepa-

ration patterns on TLC plates. Of the fourmetabolites B 1 was the most predominate andthe most toxic [ 17 ].

Further research in the years since has foundother metabolites, with two found in the milk (M1 and M 2) and urine of lactating mammals.Aflatoxins M 1 and M 2 are produced from theirrespective B aflatoxins by hydroxylation inlactating animals and are excreted in milk atthe rate of approximately 1.5% of the rate of ingested B aflatoxins. Research has alsofound that aflatoxin is most commonly foundin tree nuts, peanuts, and oilseeds includingcorn and cottonseed.

Ecology. The greatest problems associatedwith aflatoxin are in corn production and food-stuffs. These problems occur for two reasons.Corn is grown in climatic areas that give thefungi/mold the greatest opportunity for growthand dispersal, and the areas that grow cornconsume it as a main part of the diets of bothanimals and humans [ 17 ].

Aflatoxin grows best at temperatures of 80to 90 F, but can survive at temperatures aslow 40 F. The mold also needs a high mois-ture content in the host, either through kernelmoisture or in the form of rainfall. Kernelmoisture of 20% or greater is optimal, but thefungus can survive and grow in grain withmoisture content as low as 15% [ 17 ]. In corn,insect damage to developing kernels allowsentry of aflatoxigenic molds, but invasion canalso occur through the silks of developing ears.

Research conducted into the growth of afla-toxin has indicated that the fungus needs someform of associated stress in the plants for thefungus to invade. The stress may be in theform of drought that weakens the plant sys-tem, extended periods of high temperature,damage from insects or birds, high crop den-sity, or competition from weeds. All of theseconditions weaken the host or provide a meansof entry to the spores to establish a footholdin/on the host [ 17 ].

As was indicated in Chapter One, the sporesneed a means of transmittal to spread andgrow. Insects and birds can carry spores on

Chapter 3

Mycotoxins in Grain and Feed


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their bodies and when moving through a fieldcontaminate many plants. Raindrops hittinginfected plants and splashing to another planthave also been shown to be a means of trans-mittal. Poor field management in cleaning

away contaminated residual crop debris leavesthe spores in the fields to further continue thegrowth cycle in later crops.

Infection of harvested crops also occurs oncethe grain reaches storage. If corn is not cleanedand dried to adequate levels (moisture con-tent of less than 15%), the fungus will growand contaminate healthy clean grain. Hot

spots containing spores and moisture canoccur in storage bins that create a self-sus-taining environment of moisture (respiration)and heat (decaying grain) that provide primegrowing conditions for the aflatoxin fungi [1].

Figure 1. Aspergillus flavus Infection of Corn(Wicklow and Donahue, 1984)

Health Effects . Aflatoxins are both acutelyand chronically toxic in animals and humans.The disease primarily attacks the liver caus-ing necrosis, cirrhosis, and carcinomas. Noanimal has been found to be totally resistant

to the effects of aflatoxin, although suscepti-bility differs from species to species. Afla-toxin B 1 has been shown through research tobe the most potent naturally occurring carcino-gen in animals, with a very strong link to hu-man cancer incidence [ 21].

Scientists have conducted studies that haveshown a positive correlation between con-sumption and the level of intake of contami-nated food and feed, to liver cancer in Kenya,Mozambique, Uganda, and Swaziland in Af-rica, and China and Thailand in Asia. Studiesconducted in the Southeastern United Stateswhere foods that possibly contain aflatoxin aregrown also show an increase in liver cancers.While this increased incidence of cancer isstatistically apparent, there is no confirmedlaboratory link of cancer to humans, only ani-mals [ 11 ].

Aflatoxicosis refers to poisoning from the in-gestion of aflatoxins in contaminated food orfeed. It can occur from acute exposure of veryhigh doses of contaminated grain over a shortperiod of time, or from the chronic ingestionof low levels of aflatoxin over longer periodsof time [ 21].

Acute aflatoxicosis in humans is rare; how-ever, several outbreaks have been reported. In1967, twenty-six people in two farming com-

munities in Taiwan became ill with apparentfood poisoning. Nineteen were children, threeof whom died. Rice from affected householdscontained about 200 ppb of aflatoxin whichwas probably responsible for the outbreak.


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An outbreak of hepatitis in India in 1974 af-fected four hundred people, one-hundred of whom died. The outbreak was traced to corncontaining up to 15,000 ppb. It was calcu-lated the affected adults may have consumed

2 to 6 mg on a single day, implying that thelethal dose for adult humans is on the order of 10 mg.

Perhaps of greater significance to humanhealth are the immunosuppressive effects of aflatoxins. Immunosuppression can increasesusceptibility to infectious diseases, particu-larly in populations where aflatoxin ingestionis chronic, and can interfere with productionof antibodies in response to immunization inanimals and perhaps also in children.

As stated before aflatoxicosis primarily attacksthe liver but does cause other health effects.Acute symptoms include vomiting, abdomi-nal pain, pulmonary edema, convulsions,coma, and cerebral edema. Many of theseconditions can only be treated with medicalcare which is beyond that available to devel-oping or third world areas, leaving their popu-

lations at great risk.Research has shown that animals that developaflatoxin poisoning have compounding effectsthat can effect the human population aroundthem. Cows reduced milk and meat produc-tion limits the variety of diet in third worldcultures. The milk products can also havemetabolites that are passed on through the milk and milk end products including nonfat drymilk, cheese, and yogurt. In poultry, chick-

ens lay fewer eggs, and can pass on aflatoxinin the yolks of their eggs further adding to theproblem. Both the animal and human popula-tions can develop secondary immune systemproblems that leave them susceptible to fur-

ther unassociated diseases and viruses [ 17 ].

Tolerance Levels. The Food and Drug Ad-ministration (FDA) has established action lev-els for aflatoxin present in food or feed. These

limits are established by the Agency to pro-vide an adequate margin of safety to protecthuman and animal health.

seicepS ytidommoC noitcA

leveL

snamuH k liM bpp5.0

M( 1)

snamuH tpecxedoof ynA

k lim bpp02

slaminaerutammI,)yrtluopgnidulcni(

ro,slaminayriadtonsiesudnenehw

.nwonk

rehtodnanroCsniarg

bpp02

seicepSllArehtodeef laminAnottocronrocnaht

laemdeesbpp02

f eebgnideerBgnideerb,elttac

erutamro,eniwsyrtluop

rehtodnanroCsniarg

bpp001

f oeniwsgnihsiniFretaergro.sbl001

rehtodnanroCsniarg

bpp002

elttacf eebgnihsiniF rehtodnanroC

sniarg bpp003

,eniws,elttacf eeByrtluop

laemdeesnottoC bpp003

Table 1. FDA Action Levels for Aflatoxin

Detoxification. Many companies and re-searchers have tried to find a means of detoxi-

fying aflatoxin contaminated grains. Most of the treatments have been found to be eithertoo expensive to use in storage facilities or tohave side effects on the end products thatcancel the benefits of detoxification.


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Dietary supplements to strengthen the generalhealth of affected animals and humans haveshown the most promise. The addition of vi-tamins, proteins, and trace elements, alongwith inorganic absorbents added to feed, have

the most positive effects. The absorbents bindand immobilize the toxins holding them in theintestinal tract, allowing them to be eliminatedin urine and fecal matter [ 17 ].

Occupational Risks. It must be emphasizedthat there have been no definitive links be-tween aflatoxin and cancer in humans, butstudies have shown associative risks in grainhandling and processing occupations. Re-search conducted in Holland and the UnitedStates showed elevated risk of respiratory andliver cancer to those exposed to dust associ-ated with grain and flour production. Thegreatest risk appeared in flour baggers andemployees working at dump pits unloadinggrain into storage facilities. Accordingly careshould be taken when handling raw grain prod-ucts or fine end products to avoid inhalationof fine particulates [ 12].

Ergot

Pathogen. Ergot is another fungi that attackswheat, rye, barley, and oats. Claviceps

purpurea , or ergot, is the cause of the earliestrecognized human mycotoxicosis, ergotism.

Ecology. C. purpurea has three differentmeans of transmittal. Windborne ascosporescan attack the immature kernels and grow intosclerotium, purple-black hornlike structures,that replace kernels on the heads of grain. If these sclerotium mature on the stalk, they ripenand grow small club like stromata that in turngrow and release more ascospores. As the

sclerotium mature they release a substancecalled honeydew (sugary dew-like liquid) thatencourages insects to feed. This liquid con-tains small conidiophores that will attack theimmature grain heads and start the process

over and therefore spread the spores from plantto plant. The sclerotium, what are commonlycalled ergot, must be exposed to cold (36 -37 F) for several weeks before they can ger-minate. The sclerotium can overwinter in thesoil or stay dormant in storage until the properconditions exist to again start the process overagain [ 9].

The damage of ergot is two fold. It decreasesthe yields of infected crops by replacinghealthy kernels and robbing the host plant of needed nutrients. The sclerotium contain al-kaloids that can have adverse health affectson humans and animals. The grains are alsodiscounted in market value if the sclerotiumare present after harvest.

Health Effects. Animals can suffer severeblood vessel constriction that can result in drygangrene. Internal bleeding, vomiting, con-

stipation, diarrhea, and intestinal inflamma-tion are also common problems in livestock.Swine that are fed ergoty feed may abort anyfetuses that they hold.

Humans can suffer gastrointestinal distressand convulsions, abortion of fetuses, or a ne-crotic gangrenous condition of the extremi-ties if the ergot is ingested in sufficient quan-tities. Ergotism was known as St. Anthonysfire during the Middle Ages because it was

believed that pilgrimages to a shrine of St.Anthony could lead to a cure of the disease.It is likely that as pilgrims traveled to theshrine, they left areas where bread wascontaminated with ergot and traveled to areas


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where ergot was not a problem. The main er-got alkaloid, ergotamine, has vasoconstrictiveproperties that cause swollen limbs and alter-nating burning and cold sensations in fingershands and feet, hence the term fire in St.

Anthonys fire. Convulsive ergotism may alsohave been the reason for the Salem witchcrafttrials of 1692. In recent history, outbreaks of ergotism have occurred in Russia in 1926, Ire-land in 1929, France in 1953, India in 1958,and Ethiopia in 1973.

The ancient Chinese and Europeans used er-got alkaloid compounds to reduce bleedingafter childbirth and to induce abortions whenneeded. Modern science has found ways tocontrol these alkaloids. Several useful medi-cations have been developed to treat bleed-ing, muscle spasms, and migraine headaches.Two alkaloid compounds of ergot have shownremarkable effectiveness in treating migraineheadaches. Research is continuing to findmore and varied medical uses for these ergotalkaloids [ 23].

TrichothecenesPathogen . The trichothecenes are a chemi-cally related family of compounds that areproduced by fungi such as Fusarium, Tricho-derma, Myrothecium, and Stachybotrys. Thetrichothecene mycotoxins have been isolatedand found in Canada, England, Japan, SouthAfrica, and the United States. The most com-mon mycotoxins in the trichothecene familyfound in grain are DON and T-2, with ZENand Fumonisin also commonly found [ 12 ].Fusarium gramminearum , the parent fungithat produces DON, causes both GibberellaEar Rot in corn, and head scab in wheat.

Over a ten year period the Mycotoxin Labo-ratory at North Carolina State Universityfound Fusarium species of fungi in almostevery lot of corn tested. DON was detected inover 60 percent of poultry and dairy feed

tested, and ZEN was found in 15 to 20 per-cent of feeds tested.

The Fusarium species of fungi are capable of producing 70 different mycotoxins, with somespecies producing as many as 17 strains of mycotoxins simultaneously. Research is find-ing new strains of previously unknown myc-otoxins as testing methods improve and moreresearch is conducted. Fumonisin is a recentdiscovery that research indicates is very toxicto horses, but little is known of its incidenceor range [ 20].

Health Hazards. The trichothecene familyof mycotoxins affects each species of animalsin different ways. Testing done so far indi-cates that poultry has the highest resistance tothese toxins. Cattle, sheep, and goats havesome level of resistance due to their multipledigestive process, while animals of the mo-

nogastric digestive process seem to have theleast resistance to the toxins. Swine seem tobe the most sensitive, partly due to their in-creased sense of smell which leads to feed re-fusal [ 15 ].

The greatest problems associated with thesetoxins are from prolonged feed intake at lowcontamination levels. The effects depend onthe specific toxin, the duration of exposure,and the type of animal involved. All animal

species suffering from chronic toxicoses showvery good to excellent signs of improvementwhen the contaminated feed is removed. Fewlong term side effects remain with most of thisgroup of toxins if diagnosis is made quickly


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before the general health of the affectedanimals is compromised [ 12].

The processing of grain with toxins in thetrichothecene group generally does little to re-

move the toxin. Milling, baking or boilinghas only a slight effect in removing the tox-ins. Tests conducted on finished products con-taminated with these mycotoxins have shownthat 50 to 60 percent of the toxins are trans-mitted to the finished product. In some cases,such as the tempering of grain to reach a de-sired moisture level, the toxins have actuallyincreased due to the proper environmentalconditions needed for toxin production. Thetoxins can be transmitted to final productssuch as flour, bread, crackers, and cereal [ 12 ].

Deoxynivalenol (DON)

Pathogen. Fusarium graminearum is the par-ent fungi of deoxynivalenol (DON) orvomitoxin. Wheat and barley are the mostcommonly effected grain crops but the samefungus does infect corn. In the field, it shows

up as a brown discoloration at the base of bar-ley glumes, a pink to reddish mold on theglumes and kernels of the wheat heads andthe tips of the ears of corn. Spores from themold stage of the fungi can stay dormant oninfected residues left on or in the soil. Con-tamination is most severe in fields where cornfollows corn, or where corn follows wheat,especially if the previous crop was infected[24 ].

Ecology. The optimal temperature range forthe DON mold is 70 to 85 F with moisturelevels preferred to be greater than 20 percent.There are exceptions to be noted. The moldcan survive temperatures as low as 0 F for short

periods of time. This particular fungi has twodistinct growth cycles, with the mold grow-ing during the warm temperatures of daytime,while the toxins are produced during the coolertemperatures of the night [ 2].

Health Effects. The symptoms associatedwith DON poisoning are many and variedwhich sometimes leads to its misdiagnosis asa problem. At low levels of toxicoses thesymptoms may include behavioral and skinirritations, feed refusal, lack of appetite, andvomiting. In later stages, symptoms may in-clude hemorrhage and necrosis of the diges-tive tract, neural problems, suppression of theimmune system, lack of blood production inthe bone marrow and spleen, and possible re-productive problems including birth defectsand abortion [ 15, 20 ].

Table 2. FDA Advisory Levels for DON

f ossalClaminA

teiDf onoitroP mumixaM

leveLNOD

snamuHtaehwdehsiniF

,narb,ruolf (stcudorp)mreg&

mpp1

dnaf eeBelttactoldeef

4nahtredloshtnom

-ybniargdnaniarGottonstcudorp

teidf o%05deecxempp01

snekcihC-ybniargdnaniarG

ottonstcudorpteidf o%05deecxe

mpp01

eniwS-ybniargdnaniarG

ottonstcudorpteidf o%02deecxe

mpp5

rehtollAslamina

-ybniargdnaniarGottonstcudorp

teidf o%04deecxempp5


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As stated earlier, DON stays in end feed prod-ucts even after processing. In 1995, 16,000tons of dog food was produced using wheatby-products (most probably dust). After itreached the consumer level it was found to

contain DON in excess of 30 ppm. The prod-ucts were recalled costing the company inquestion a loss of approximately $20 million[16 ].

Tolerance Levels. Canada has set action lev-els for DON in grain and finished products,while the United States has set only advisorylevels. Canadas tolerance levels are set at lessthan 2 ppm in wheat for human consumption,and less than 1 ppm for wheat destined foruse in infant food products.

The United States has set their advisory lev-els at less than 2 ppm for wheat destined forhuman consumption, and less than 5 ppm formost animal feed products [ 12].

Zearalenone (ZEN)

Pathogen. Zearalenone is very similar todeoxynivalenol (DON) in most aspects with afew exceptions.

Ecology. The growing conditions of ZEN arevery comparable to DON, with the optimaltemperature range of 65 to 85 F. A drop intemperature during growth also stimulates theproduction of toxins [ 2].

The moisture content required by ZEN is alsosimilar to DON at 20 percent or greater. Butif the moisture content during growth dropsbelow 15 percent the production of toxins ishalted. This is one of the reasons that corn

for storage must be dried to moisture levelsless than 15 percent [ 24].

Health Effects . The greatest difference be-tween ZEN and DON is the way the toxin acts

in animals. ZEN mimics the hormone estro-gen in the way it effects animal tissue. Swineare the most sensitive to its effects with levelsof 1 ppm causing feed refusal. Continued con-sumption of contaminated grain will causeestrogenism (health problems related to thereproductive system). These effects includeswelling of the reproductive organs includingthe genital and mammary glands, interruptionof the reproductive cycles, birth defects, andatrophy of the ovaries and testes. In male ani-mals, feminization occurs with enlargementof the mammary glands and loss of sex drive[20, 2 ].

Poultry show little or no effects from ZEN con-sumption. Cattle also show very little effectexcept in cases of prolonged consumption of high levels (greater than 15 ppm) of ZEN. Theeffects produced are reduced milk production,swollen reproductive organs, and in some

cases infertility. The ZEN is passed throughthe system with very little absorption shownin milk, urine, or body tissues [ 2].

Tolerance Levels. The FDA has issued noadvisory levels for zearalenone recommend-ing only that the levels of concern for DONbe observed (See above). Levels of as little as0.1 ppm to 5 ppm have been shown to causereproductive problems in swine so great careshould be used when feeding wheat that is

possibly contaminated to pigs [ 24 ].


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Fumonisin

Pathogen. Fumonisin moniliforme is the par-ent fungi species of fumonisin. This funguscauses Fusarium Ear Rot in corn which is themost common disease of corn in the UnitedStates Midwest region. Testing of corn fieldshas shown that over 90 percent of fields areaffected by this fungi in one of its variousstrains [ 24 ].

The mold appears on the corn ears as a cot-tony white to light grey filaments between thecorn kernels. As the mold progresses the ker-nels will turn grey to light brown.

The fumonisin toxin can grow in the kernelseven with no apparent outward signs of mold.Testing of the grain is the only positive meansof verifying whether fumonisin is present ornot [24].

Ecology. Growing conditions vary widely.The temperature and moisture ranges are sowide spread as to include most of the North-ern and Southern Hemispheres. The one com-

mon factor associated with fumonisin is thathigher incidence of infections seem to occurafter periods of drought which stress the plantsimmune system [ 24].

Fumonisin causes the corn kernels to becomebrittle and crack more frequently than is nor-mal. The more the grain is handled the morecracking and breaking occurs, giving the fungimore host material to grow on. For this rea-son corn screenings should be very suspectwhen used as feed, especially in horses. Test-ing has shown that screenings contain a higherlevel of fumonisin toxin (and mycotoxins ingeneral) than the whole grain product [ 24 ].

Health Effects. Fumonisin is one of the my-cotoxins that has only recently been discov-ered and has been little studied. The relatedhealth effects have shown few effects in hu-mans and most animals other than swine and

horses. While in depth research is lacking,fumonisin has shown a high degree of toxic-ity in preliminary studies conducted in horses.Swine have shown little or no effects with theonly preliminary symptoms to be possible res-piratory problems and possible links to theliver and kidneys. [ 20].

Toxin levels of as low as 5 ppm have showndirect links in horses with symptoms whichinclude: disorientation, walking/agitation, de-rangement, colic, head pressing, blindness,and death. The toxin seems to attack the liverand kidneys, which is similar to other myc-otoxins except in the severity. Fumonisin hasalso been linked to equine leukoencephal-omalicia, also known as Blind Staggers (acomplete breakdown of the neural system inthe brain) which has a high mortality rate [ 20].

Tolerance Levels. Currently there are no ac-

tion or advisory levels in place by the FDAbecause so little is known about the effects.Industry levels have recommended levels tobe no higher than 5 ppm in horses, 10 ppmfor swine, and 50 ppm for cattle [ 24].

Nivalenol (NIV)

Pathogen. Nivalenol is produced by theFusarium nivale fungi and has also only re-cently been isolated. Little is known of itsgrowth cycle or habitat range. Studies haveshown it to be much rarer in occurrence andhas only been found in a few samples of bar-ley, wheat, wheat flour, and rice [ 12 ].


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Health Effects. Though little actual test datahas been produced, the results so far cause sci-entists to be extremely cautious. Preliminarytesting shows that NIV is thought to be 10times more potent than DON. If the advisory

level for Don is used as a guide for toxicity,NIV would have an advisory level of only 0.2ppm [ 12].

Ochratoxin

Pathogen. Ochratoxins were initially associ-ated with Aspergillus ochraceus but are pro-duced primarily by Penicillium verrucosum .

Ecology. A. ochraceus is widely distributedin dried foods such as peanuts, pecans, beans,dried fruit, and dried fish. Ochratoxin con-tamination of foods is of greatest concern inScandinavia and the Baltic states where it isproduced by Penicillium verrucosum . It isfound in barley and wheat crops infected inthe field or in storage crops used for both breadmaking and animal feeds.

Health Effects. Ochratoxin is the most im-portant toxin produced by a Penicillium spe-cies. It can cause listlessness, huddling, diar-rhea, tremors, and other neural abnormalitiesin poultry and has been associated with kid-ney disease in swine in Scandinavia and north-ern Europe.

Because ochratoxin A is fat soluble and notreadily excreted, it accumulates in the fat of affected animals and from there is ingestedby humans eating pork. A second source isbread made from infected barley or wheat.

T-2

Pathogen. Fusarium tricinctum and somestrains of F. roseum produce T-2 . T-2 hasbeen found in corn in the field, silage, andprepared feeds made with corn.

Health Effects. During WWII, a very severehuman disease occurred in the former SovietUnion. Alimentary toxic aleukia (ATA) is be-lieved to have been caused by T-2 and HT-2in grain left to overwinter in the field. Whenthis grain was consumed, severe mycotoxico-sis occurred. ATA results in a burning sensa-tion in the mouth, tongue, esophagus, and

stomach. Eventually the blood making capac-ity of the bone marrow is destroyed and ane-mia develops. In the final stages hemorrhag-ing of the nose, gums, stomach, and intestinesdevelops and the mortality rate is high. In poul-try, T-2 may produce lesions at the edges of the beaks, abnormal feathering, reduced eggproduction, eggs with thin shells, reducedbody weight gain, and mortality.

Cyclopiazonic Acid (CPA)

Pathogen. CPA is produced by A. flavus andseveral Penicillium species. It has been foundin corn and peanuts in Georgia. The principlePenicillium species producing CPA, P. com-mune , is a cause of cheese spoilage aroundthe world.

Health Effects. CPA is a highly toxic com-

pound that causes fatty degeneration and he-patic cell necrosis in the liver and kidneys of domestic animals. Chickens are particularlysusceptible. When the compound is injectedinto experimental animals, central nervous


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dysfunction occurs and high doses can resultin death. CPA and aflatoxin may act syner-gistically when consumed together by animals.

CitrininPathogen. Citrinin is primarily a metaboliteof Penicillium citrinum but is also producedby P. expansum and P. verrucosum. Thesethree species are the most commonly occur-ring penicillia, and so citrinin is probably themost widely produced Penicillium toxin.

Ecology. Citrinin has been isolated from al-most every kind of food surveyed for fungi.The most common sources are cereals suchas rice, wheat, and corn, milled grains, andflour.

Health Effects. Citrinin is a kidney toxinwhich has been associated with mycotoxicosesin swine, horses, dogs, and poultry. No toxiceffects to humans have been noted when cit-rinin is ingested in the absence of other tox-ins, however, there is a possibility of syner-gism if ingested with other toxins.


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The first step in mycotoxin analysis is obtain-ing a representative portion. Great care shouldbe taken when sampling, since sampling er-ror is often the greatest source of variance inthe analytical procedure.

The sampling and testing of grain crops in-fected with mycotoxins presents several prob-lems. Unlike protein or moisture content incorn or wheat, where every kernel tested hassome level of content (a uniform distribution),mycotoxin content does not occur in everykernel. In the extreme it may only occur in oron a few ears or heads in an entire field (annonuniform distribution). The greater the ex-tent of contamination the more likely that thedistribution will be uniform and test resultsaccurate.

Sample Size. There is a smaller sampling er-ror associated with processed commodities,such as flour, than is associated with wholeseeds. This is because of the smaller particlesize which increases the sample population(number of possibly infected particles), and

Chapter 4

Sampling

12 13 12 14

13 13 14 12

15 11 12 12

13 14 11 9

13 12 12 13

Protein0 0 0 0

0 0 0 0

0 0 0 0

0 200 0 0

0 0 0 0

Aflatoxin

Protein Avg. 13% Aflatoxin Avg . 10 ppb

the greater degree of mixing associated withthe production process.

There is a higher incidence of error in wholegrains because the number of infected particlesmay be as little as 0.1 percent of the total popu-lation of the sample. When the fact that asingle kernel of corn was tested and found tocontain aflatoxin at a level of 400,000 ppb, itis apparent why the accuracy of the represen-tative sample is so critical [ 6 ].

Each pound of corn contains approximately1,530 kernels. Ten pounds of corn containsabout 15,300 kernels of corn. With the greaternumber of kernels in a larger sample comes ahigher probability of correct test results forthat sample.

GIPSA has determined that the optimum afla-

toxin sample size for corn is a minimum of 10pounds. This sample is then ground and asubsample of 500 grams obtained. Thissubsample is then mixed and a 50 gram sampleobtained to be tested.

GIPSA has not established an optimum afla-toxin sample size for wheat, barley, sorghumand other grains. Currently the ten poundsample size is used by default for all grainswhen testing for aflatoxin.

GIPSA established reduced sample sizes fordomestic shipments of corn by truck and rail.The smaller sample sizes were adopted in re-sponse to industry concerns that the ten pound


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sample size would significantly increase thecost of inspection. The GIPSA minimumsample sizes are listed in Table 3.

Table 3. GIPSA Minimum Sample Sizes

reirraC elpmaSmuminiM

sk curT sdnuopowT

)smarg809(

sracliaR sdnuopeerhT

)smarg2631(

segraB / stolbuS sdnuopneT

)smarg0454(

selpmaSdettimbuS sdnuopneT

dednemmocer

It should be noted that using the reducedsample sizes significantly increases the sam-pling variability. Table 4 illustrates how re-ducing the sample size affects sample vari-ability.

Table 4. Truck containing 20 ppb aflatoxincontaminated corn (Romer Labs 1995).

eziSelpmaS slenreK ytilibairaV

)bpp(

.sbl01 000,03 4.82-6.11

.sbl0.5 000,51 9.13-1.8

.sbl5.2 005,7 8.83-2.3

.sbl0.1 000,3 9.64-1

However, a 1998 GIPSA study of DON con-taminated barley have shown that increasingthe sample size does not appear to significantlydecrease the variability of DON results in bar-ley. This does not mean that sample size is

unimportant for DON analysis of barley. Forsome sufficiently small sample, size would be-come a significant factor.

The sample size required by GIPSA for bar-ley and wheat is a minimum of 200 grams andpreferably larger. This sample is then groundand a 50 gram subsample obtained. The ef-fect of sample sizes smaller than 100 grams isunknown, but larger sample sizes do not ap-pear to appreciably improve the precision of DON test results in barley. Grains with largerkernel size such as corn would require largersample sizes to maintain the same level of uniformity.

Sampling Variability. The next variable thatenters the picture is sampling variability. Toensure that the sample that is tested is accu-rate, proper sampling techniques must be usedto obtain a representative sample. A boot

sample from the exposed layer of grain in ahopper car or truck, or a bucket sample as atruck or railcar is unloaded does not give arepresentative sample of the lot as a whole.

In fact, nearly 90% of the error associated withaflatoxin testing can be attributed to how theoriginal sample was obtained. This is due toonly one to three percent of the kernels in acontaminated lot containing mycotoxin, andthese contaminated kernels are not generally

distributed evenly in the lot [20]. A studyconducted by Michigan State University foundthat the variability of DON measurements intrucks of newly harvested soft red winter wheatwas significantly higher if less than four


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probes were taken from the lot [8].

Representative Sample. Obtaining a repre-sentative sample from a lot of grain is an im-portant and essential part of mycotoxin analy-

sis. If the sample is not representative theanalysis result will not represent the true qual-ity of the lot. In order for a sample to be con-sidered representative, it must be:

1. Obtained with equipment/proceduresdesigned to obtain sample from all areasof the lot;

2. Of appropriate size;3. Adequately identified;4. Handled in such a way as to maintain

representativeness.

Sampling Methods. Of all the sampling de-vices available, the mechanical sampler ob-tains the most representative sample from lotsof grain. They are powered either pneumati-cally, electrically, or hydraulically.

The diverter-type (D/T) mechanical system isused for commodities with large particle size

such as whole grain. Even though D/Ts varyin design, all operate on the same principle.

Installed at the end of a conveyor belt or withina spout, they draw their sample by periodi-cally moving a pelican (named for its resem-blance to the beak of a pelican) through theentire grain stream.

Figure 2. Diverter-Type sampler installed in spout.

Figure 3. Diverter-type mechanical samplingsystem used for whole grain.

The frequency of these cuts is regulated bytimer controls. After the grain enters the pri-mary sampler, it flows through a tube into asecondary sampler. The secondary sampler


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reduces the size of the sample. From the sec-ondary sampler, the sample flows to a collec-tion box or sample bucket.

Point-type (P/T) mechanical sampling systems

are commonly used for powdered commodi-ties. These commodies are more homoge-neous than whole grain and have less particlesegregation. They do not use a pelican to com-pletely cut across the stream of commoditythrough a spout. Instead they use a tube witha hole or slot and an auger delivery system.

A large percentage of grain, as it travels fromthe farm to the final consumer, is sampled witha probe sometimes referred to as a trier. Theprobe is the only sampling method approvedby GIPSA for stationary lots. If probe sam-pling is performed correctly, the samplesdrawn are considered representative.

Figure 4. Point-type mechanical sampling systemused for powdered commodities.

Figure 5. Grain probe or trier

Probes are constructed of brass or aluminumand come in various sizes with standardlegnths of 5, 6, 8, 10, and 12 feet. The type of carrier dictates which probe length is used.Probes consist of two tubes, one inside the

other.

GIPSA approved grain probes are 13/8 inchesin diameter (outer tube). The inner tube isdivided into compartments. The outer tubehas slots which match the compartment open-ings of the inner tube. When the tubes arealigned, grain may enter into or be emptiedfrom the compartments of the probe.

The lengths of double-tube compartmentedprobes approved by GIPSA for sampling lotsof bulk grain can be found in Table 5.

Table 5. GIPSA approved probe sizes for samplingstationary lots of grain.

reirraC )teef (htgneL

reliart / k curtdeb-talF 6ro5

reliartmottobreppoH 01ro,8,6

racxoB 6

racreppoH 21ro01

egraB 21

Sampling patterns. GIPSA has establisheda sampling pattern for each type of carrier.Each lot should be probed in as many addi-tional locations as necessary to assure that thesample is the required size and representativeof the lot. Additional probes should be drawnin a balanced manner. For example, one com-partment of hopper car should not be probed


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twice unless the other compartments are alsoprobed twice, regardless of the amount of grainin any one compartment or the amount of ad-ditional sample needed.

The following diagrams indicate the standardsampling patterns. Insert the probe at thepoints marked, with the tip of the probe angledten degrees in the direction of the arrow. Whentwo arrows are shown, the tip of the probe maybe pointed in either of the indicated directionsat the samplers discretion.

Figure 6. Seven probe pattern for flat-bottomtrucks or trailers containing grain more than fourfeet deep.

Figure 7. Nine probe pattern for flat-bottom trucksor trailers containing grain less than four feet deep.

Figure 8. Hopper bottom trailers or containers.

Figure 9. Boxcar

Figure 10. Hopper car

Figure 11. Roll-top barge


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Figure 12. Flat-top barge

Sacked Grain. When grain is sampled fromsacks, a double-tubed, compartmented grainprobe (4 feet minimum length) should be used.Stand the sacks up on end and insert the probeinto the top corner of the sack. Move the probediagaonally through the sack until the probetouches the bottom of the opposite corner.


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Chapter 5

Analytical Procedures

Laboratory Safety. In the previous discus-sion of the toxicity of the mycotoxins it shouldbe apparent that the toxins attack the systemsof animals and humans in many different andharmful ways. Caution should be the guidewhenever dealing with crops directly from thefield and in the laboratory.

Mycotoxins can cause three types of illness:

Infection. Invasion of living tissue by micro-organisms.

Allergies. Hypersensitivity of tissues to bac-teria and fungal agents.

Toxicoses. Chronic or acute disease from ex-posure to mycotoxins [ 19 ].

All samples that are being prepared for analy-

sis should be treated as being toxic untilproven otherwise. Personal protective equip-ment should be used whenever handlingsamples in the lab or when dealing with haz-ardous chemicals. This should include the useof non-permeable gloves, safety glasses, anddust masks whenever possible [19].

Dust creation should be kept to minimumwhenever possible. Laboratories should neverbe dry swept but instead vacuumed with vacu-ums equipped with high efficiency particulatefilters. Grinding of samples should occur ei-ther in negative pressure rooms or in enclosedareas with exhaust hoods that will remove asmuch of the fine particulates as possible [19 ].

Grinding. Once the sample is obtained it isonce again enlarged by grinding to a fineparticle size. The grinding opens up infectedkernels and distributes the particles through-out the sample giving an increased chance of detecting contaminated particles. To increasethe probability of finding contaminated grain,more samples could be obtained to increasethe sample size yet further. By doubling thesample size the sampling variance is reducedby 50 percent. But the larger the sample themore cumbersome the process becomes. If aship lot of corn is comprised of 20 sublots, asingle sample of 200 pounds could be groundand analyzed, but it is statistically better toanalyze 20 samples of 10 pounds each to bet-ter use resources and obtain more accurateresults [ 6 ].

Various types of grinders exist to handle dif-

ferent products and the varying degrees of hu-midity they are subjected to in storage. Somegrinders allow the screen size to be changed,others provide removable disc heads and vari-ous hammer speeds. Grinders such as theRomer mill, Bunn Model G3, VikingHammermill, Falling Number Mill, and UDYgrinder are a few examples.

Care should be taken to always use a cleangrinding system. More care must be takenafter grinding a hot sample than after aclean one. Some labs run a clean samplebetween lots for the purpose of cleaning themill. Other labs run a few grams of the nextsample and discard it to purge the mill.


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These practices are no guarantee that crosscontamination will be eliminated. Only if thegrinder can be completely opened up andcleaned can cross contamination between suc-cessive samples be avoided. The use of a

vacuum can make this procedure effortless.

Attaching a plastic bag to the outlet spout of the grinder will capture all of the sample with-out releasing dust into the air. The Romer millis capable of automatically subdividing largeten pound samples into a representative 500gram subsample. When using other grindersa riffle divider must be used to reduce tenpound samples to 500 grams. Once the 500gram sample is obtained, blend the sample bylifting or rolling the ends of the bag to theopposite side and repeating at least ten times.Allow the dust to settle before opening the bag.The 50 gram portion can now be removed andweighed.

Blacklighting . Screening of corn for pos-sible aflatoxin contamination using a black light was popular in the 1970s. In spite of the widespread use of black lighting, research

has shown that the technique detects materi-als that are not mycotoxins. In addition, my-cotoxins such as DON do not fluoresce; there-fore blacklighting is inappropriate. The blacklight should no longer be used for any typeof mycotoxin screening [20].

Minicolumn. The minicolumn is a small col-umn containing silica gel and florisil to whichsample extracts are applied for detection of aflatoxin. Minicolumns were very popular

until quick-test kits became available over thelast few years. Although capable of givinggood results under the proper conditions, theminicolumn is no longer a GIPSA approvedofficial method for mycotoxin analysis..

TLC/HPLC. Since 1990 the officially ap-proved method of testing by the Associationof Official Analytical Chemists (AOAC) formycotoxins, and aflatoxins in particular, hasbeen thin-layer chromatography. In recent

years High Pressure Liquid Chromatography(HPLC) has replaced TLC. TLC is no longera GIPSA-approved official method for myc-otoxin analysis.

Quick Tests. Newer processes have been de-veloped for quick tests that will give resultsin a shorter period of time with less use of hazardous or toxic chemicals and procedures.Four tests that will be covered in more detailthat use these methods are the Neogen Veratox,Romer AccuTox, Vicam Aflatest, and RomerFluoroquant.

The Neogen Veratox and Romer AccuToxmethods use direct competitive EnzymeLinked Immunosorbent Assay (ELISA) tech-nology. Vicams Aflatest procedure and RomerFluoroquant process use fluorescence technol-ogy.

All of these tests are quicker than HPLC, withaccuracy that is acceptable in the grain pro-cessing and feed industries. HPLC is used asa reference method to gauge the accuracy of the other tests and in laboratory use wheregreater accuracy is needed.

NEOGEN (Aflatoxin and DON)

Neogens Veratox (DON) and Veratox-AST (Aflatoxin) use ELISA technology. An-tibodies specific for a mycotoxin are adheredto the bottom of a microwell.


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A solution of mycotoxin, chemically conju-gated to an enzyme, is provided with the kit.A sample to be tested for mycotoxin is groundand extracted. The extract is then filtered andmixed with a fixed amount of themycotoxin-enzyme conjugate solution in amixing well.

Figure 13. Neogen Microwell.

Figure 14. Mixing well containing extract andconjugate.

A portion of the mixture is then transferred toa microwell. The mycotoxin from the ex-tracted sample and mycotoxin-enzyme con-

jugate then compete for binding to the anti-bodies in the microwell.

Figure 15. Free toxin and conjugate compete forbinding sites in microwell containing antibodies.

The assay procedure measures how much of the conjugate actually binds to the antibodiesby first thoroughly washing the microwell thenadding a colorless substrate.

Figure 16. After washing substrate is added.

Figure 17. The conjugated enyme and substratereact to form a blue color.

The enzyme present in the microwell converts

the substrate to a blue colored product; themore mycotoxin-enzyme conjugate in themicrowell, the more intense the blue color.Because samples with high mycotoxin willresult in less binding of the mycotoxin- en-zyme conjugate, positive samples will belighter blue. Quantitative measurements areobtained by measuring the intensity of thecolor with an optical density reader.

ROMER (Aflatoxin)

Romer Labs FluoroQuant test for aflatoxinuses fluorescence technology. A sample to betested is ground and extracted with methanol/


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water (80/20). The extract is filtered and aportion of the filtrate is placed in the top of aUniSep 2001 column. An equal portion of diluent is added and the solutions are thor-oughly mixed by shaking. The column is

placed into a cuvette and a syringe barrel at-tached. The extract is slowly pushed throughthe column.

A portion of the purified extract is transferredto a clean pipet and a developer reagent isadded to enhance the fluoresence of the afla-toxins. The solution is mixed by vortexingand the sample is read in a fluorometer.

Figure 18. The filtrate and diluent are placed intoa UniSep column, mixed, and forced through thecolumn

ROMER (Deoxynivalenol)

Romer Labs AccuTox test for deoxynivalenolis an ELISA method. Antibodies specific fora mycotoxin are adhered to the bottom of a

tube.

Figure 19. Romer Antibody Tube.

A sample to be tested for DON is ground, ex-tracted with distilled water, and filtered. Asolution of DON, chemically conjugated toan enzyme, is provided with the kit. The fil-tered extract is mixed with an equal amountof the mycotoxin-enzyme conjugate in an

antibody tube.

Figure 20. Free toxin and conjugate compete forbinding sites in microwell containing antibodies.


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After the toxin and conjugate have had timeto attach to the antibodies, the remaining so-lution is discarded and the tube rinsed withdistilled water.

After tapping the tube on a paper towel to re-move the water, substrate solution is added tothe tube. The conjugated enzyme present con-verts the substrate to a blue colored product;the more conjugate in the tube, the more in-tense the color.

Figure 21. After washing. substrate is added whichreacts with the conjugated enzyme to form a bluecolor.

After allowing the color change to develop forfive minutes, stop solution is added. Thisstops the reaction and changes the color of the solution to yellow. Quantitative measure-ments are obtained by measuring the inten-

Figure 22. The solution turns yellow when thestop solution is added.

sity of the color with a Hach spectrophotom-eter. Because samples with high DON levelswill result in less binding of the conjugate,positive samples will be lighter color.

VICAM (Aflatoxin)

Vicams columns use immuno-affinity chro-matography technology. The columns con-tain antibodies specific for the mycotoxinschemically fused to beads.

Figure 23. The aflatest column contains antibod-ies fused to glass beads.

A sample is ground and extracted with a

methanol/water solution. The extract is thendiluted and run through the affinity column.

Figure 24. The filtered and diluted extract is runthrough the affinity column.


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The mycotoxin sticks by binding to the anti-body beads. Other materials in the extract donot stick and are washed off the column.

Figure 25. Impurities do not stick to the antibod-

ies and are washed off.

The mycotoxin is then removed from the col-umn using methanol.

Figure 26. The toxin is removed with a methanolwash.

A developer containing bromine is added toenhance the fluorescence by making a deriva-tive of the mycotoxin. The level of mycotoxinis measured with a fluorometer.


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GIPSA Approved Mycotoxin MethodsApril 1999

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NEOGEN 620 Lesher Place, Lansing, MI 48912, (800)-234-5333

VICAM, L.P. 313 Pleasant Street, Watertown, MA 02172, (800)-338-4381

Romer Labs 1301 Stylemaster Drive, Union, MO 63084, (314) 583-8600

International P.O. Box 799, St. Joseph, MI 49085, (616) 428-8400DiagnosticSystems Corp.

EDITEK 1238 Anthony Road, Burlington, NC 27215, (800) 334-1116

Diagnostix 5730 Coopers Avenue, Unit #27, Mississauga, Ontario, Canada L4Z2E9,(905) 890-6023


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Aflatoxicosis A poisoning that results from ingestion of aflatoxins in contaminated foodor feed.

Ascospores A sexual spore formed in an ascus.

Ascus Saclike structure in which ascospores are borne.

Blight General term for sudden, severe, and extensive spotting, discoloration,wilting, or destruction of leaves, flowers, stems, or the entire plant.

Carcinogen A substance or agent producing or inciting cancer.

Carcinoma A new growth or malignant tumor enclosing cells in connective tissue.

Cirrhosis A chronic disease of the liver characterized by progressive destruction andregeneration of liver cells, ultimately resulting in liver failure and death.

Coleoptiles Ephemeral, nonpigmented tissue sheathing the first true leaf of a grass(maize) seedling.

Clums Stem of a grass plant.

Cultivars Cultivated variety.

Embryo Seed germ; the rudimentary plant within a seed.

Florets Small flower enclosed in a spikelet.

Fungicide Chemical or physical agent that kills or inhibits the growth of fungi.

Fungus (fungi) Organism having no chlorophyll, reproduces by sexual or asexual sporesand not by fission, and, generally, a mycelium with well-marked nuclei.

Germination Begin growth as of a seed, spore, sclerotium, or other reproductive body.

Glumes Empty bract at the base of a spikelet.

Glossary(Derived from underlined words in text)


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Host Living plant attacked by (or harboring) a living parasite and from whichthe invader is obtaining part or all of its nourishment.

Hybrids Offspring of two individuals of different genetic character.

Hyphae A tubular, threadlike filament of fungal mycelium.

Inoculum Spores or other diseased material that may cause infection.

Lesions Well-marked but localized diseased area; a wound.

Metabolite A product of the chemical changes in living cells by which energy isprovided for vital activities and processes and new material is assimilated.

Mycelium Mass of hyphae constituting the body (thallus) of a fungus.

Mycotoxicoses Literally, fungus poisonings; current usage limited to poisoning of peopleand animals by various food and feed products contaminated (and sometimes rendered carcinogenic) by toxin-producing fungi.

Necrosis Death of plant or animal cells, usually resulting in tissue turning dark;commonly a symptom of fungus, nematode, virus, or bacterial infection.

Pathogen Organism or agent that causes disease in another organism.

Pericarp Outer layer of a seed or fruit.

Perithecia A small fruiting body in certain fungi, containing ascospores.

Sclerotia Hard, frequently rounded, and usually darkly pigmented resting body of afungus composed of a mass of special hyphae cells. The structure mayremain dormant for long periods. Sclerotia germinate upon return of favorable conditions to produce stroma, fruiting bodies and mycelium.

Sori Compact fruiting structure of rust and smut fungi.

Spikelet Spike appendage comprised of glumes and florets; unit of inflorescences

in grasses.

Spore One-to-many-celled reproductive body in fungi and lower plants that candevelop into a new plant.


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Subcrowninternode Short, culm-like connection between the crown and seed roots of wheat.

Teliospores Thick-walled resting spore of rust and smut fungi that germinates to forma basidium.

Toxin A poisonous substance, having a protein structure, that is secreted bycertain organisms and is capable of causing toxicosis when introducedinto the body tissue. Toxins are also capable of inducing an antitoxin.

Trichothecene A group of chemically related compounds produced by fungi such asFusarium.


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1. Black, Kevin. Aflatoxins in Corn (Dec. 1996): 2 pag. Online. Internet. 20, Feb. 1997

2. Cheeke, Peter R. , Lee R. Shull . ed. Natural Toxicants in Feeds and Poisonous Plants.Westport: AVI Publishing 1985.

3. Chen, Yuan-Kuo . Zearalenone CU Toxic Plant Pages (nd): 2 pag. Online. Internet.

4. Christensen, CM., C.J. Mirocha, R.A. Meronuck. Molds, Mycotoxins, and Mycotoxi-coses Cereal Foods World Vol. 22 No. 10 (Oct 1977): 513-520.

5. Cook, James R., Roger J. Veseth . Wheat Health Management St. Paul: APS Press 1991.

6. Council For Agricultural Science and Technology . Mycotoxins: Economic and Health

Risks. Iowa: Task Force Report No. 116, 1989.

7. Grainaissance, Inc . What is Amazake (nd): 4 pag. Online. Internet. 19, Feb. 1997.

8. Hart, L.P. Variability of Vomitoxin in Truckloads of Wheat in a Wheat ScabEpidemicYearPlant Disease Volume 82 No.6 June 1998

9. Karow, Russ . Ergot in Cereal Grains - Cause, Hazards and Control OSU ExtensionService (nd): 2 pag. Online. Internet 19, Feb. 1997.

10. Karow, Russ., Dick Smiley . Karnal Bunt Wheat Diseases (nd) 3 pag. Online. Internet.4, Feb. 1997.

11. Known Carcinogen: Aflatoxin [Cas. No. 1402-68-2] (nd): 2 pag. Online. Internet.20, Feb. 1997.

12. Krogh, Palle. ed. Mycotoxins in Food. San Diego: Academic Press Limited 1987.

13. Mathre, D.E. ed. Compendium of Barley Diseases St. Paul: APS Press 1991.

14. McMullen, Marcia P., Robert W. Stack . Head Blight (Scab) of Small Grains NDSUExtension Service pp-804, (nd): 6 pag. Online. Internet. 6, Feb. 1997.

15. Miller, J.D., H.L. Trenholm ed. Mycotoxins in grain: Compounds Other Than Aflatoxin.St. Paul: Eagan Press 1994.

References


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16. Mycotoxins in Feed Grains Dept. of Botany & Plant Pathology, Purdue University(July 1996)

17. Saad, Nabil., Aflatoxins: Occurrence and Health Risks (nd): 7 pag. Online. Internet.20, Feb. 1997.

18. Shurtleff, Malcom C . ed. Compendium of Corn Diseases 2 ndedition. St. Paul: APS Press1992.

19. Trucksess, Mary W., John L. Richard . Labortory Safety Considerations in the Handlingof Natural Toxins. Fort Collins: Alaken, Inc. 1992.

20. Understanding and Coping With Effects of Mycotoxins in Livestock Feed and For-age North Carolina Extension Service (nd)

21. US Food and Drug Administration Center for Food Safety and Applied Nutrition.Aflatoxin Foodborne Pathogenic Microorganisms and Natural Toxins [Bad Bug Book](1992): 2 pag. Online. Internet. 4, Feb. 1997.

22. Volk, Tom. Fungus of the month of February 1997 (Feb. 1997): 3 pag. Online. Internet.26, Feb. 1997.

23. Wiese, M.V. ed. Compedium of Wheat Diseases. 2 nd edition. St. Paul: APS Press 1991.

24. Woloshuk, Charles P . Dept. of Botany & Plant Pathology, Purdue University Mycotoxinsand Mycotoxin Test Kits, Cooperative Extension Service, Corn Diseases BP-47 (July 1994):

12 pag. Online. Internet. 10, Feb. 1997.25. Woloshuk, Charles P . Dept. of Botany & Plant Pathology, Purdue University Vom

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Mycotoxinas en Cereales