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Mycotoxins and Mycotoxicosis in Swine

Carlos A. Mallmann Paulo Dilkin

Translated by Gabriela Zaviezo, M. A. Edited by Douglas Zaviezo, Ph. D. From the original Portuguese version published in 2007 and updated by the authors

2 PRESENTATION Special Nutrients, a U.S. based company, as a sponsor of this book and as a world pioneer in the applied science of mycotoxin detoxification, would like to take this opportunity to thank the authors, Dr. Carlos Agusto Mallmann and Dr. Paulo Dilkin of the Department of Preventive Veterinary Medicine at the Universidade Federal de Santa Maria, RS, Brazil, for allowing us to translate this excellent book, which provides the technical community around the world knowledge regarding mycotoxins, their dangerous effects, and technical recommendations to maintain the optimal productivity of swine. It is our pleasure to briefly introduce the authors of this book. Carlos A. Mallmann Carlos A. Mallmann received his Doctor in Veterinary Medicine and Master of Science degrees from the Universidade Federal de Santa Maria (UFSM), RS, Brazil. He received his Doctorate in mycotoxicology from the Free University of Berlin and the Superior School of Veterinary Medicine of Hannover, Germany. He is currently professor of Public Health at the UFSM and director of the Laboratory of Mycotoxicological Analyses (LAMIC). Dr. Mallmann is also a consultant for official institutions such as FAO/ONU, and for some of the most important animal nutrition and feed manufacturing companies in Brazil and other countries. Paulo Dilkin Paulo Dilkin is Doctor in Veterinary Medicine, specialized in animal toxicology. He has a Masters in preventive veterinary medicine and Doctorate in Microbiology (mycotoxicology). He is currently a professor in the Department of Preventive Veterinary Medicine at the Universidade Federal de Santa Maria (UFSM), RS, Brazil. Dr. Dilkin is a scientific adviser for LAMIC/UFSM, developing analytical procedures, experimental mycotoxicological animal tests and production of mycotoxin standards. He is also a legal expert in mycotoxin involvements, and a consultant in implementing quality control programs for monitoring mycotoxins in feed and feed ingredients, as well as the development and utilization of antimycotoxin additives.

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Index

Foreword ................................................................................................................. Mycotoxins................................................................................................................

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Aflatoxins.................................................................................................................. 20 Zearalenone.............................................................................................................. 55 Fumonisins................................................................................................................ 78 Trichothecenes.......................................................................................................... 98 Ochratoxin A............................................................................................................ 117 Other mycotoxicosis in swine.................................................................................. 136 Modern techniques in managing and controlling mycotoxins............................... 159

4 FOREWORD Mycotoxins constitute a highly dynamic field of study; everyday new information is generated, creating greater challenges. Therefore, we are constantly emerged in this fascinating and dynamic professional field. We believe that this book could be a significant contribution to the swine industry, which has already reached a high level of productivity and performance through new technologies in the areas of genetics, nutrition, management and biosecurity. Mycotoxins are becoming increasingly more important because of how they limit improvements in efficiency and productivity that have already been implemented. Therefore, we think that mycotoxicology will be one of the strategic areas in the near future, not only in Brazil, but also around the world, because of mycotoxins’ repercussions on the deleterious effects on the safety of human and animal nutrition and its economical implications. This book was written using practical knowledge acquired during our professional lives, but was fundamentally based on results of research published by the international scientific community. However, this is not a finished project, since not all the knowledge generated until today could be presented in this book. There is also new research published on this topic every day. Nonetheless, we hope to present some important basic knowledge, addressed mainly to veterinarians, researchers, students, professionals, and swine producers. We begin this book by defining some basic concepts concerning mycotoxicological dynamics. Then, we detail concepts concerning aflatoxins, zearalenone, fumonisins, trichothecenes, ochratoxin A, and other less prevalent and less well known mycotoxins. Finally, we highlight the actions that should be taken or the way to manage mycotoxins, proposing different tools that are approved and widely used in mycotoxicological monitoring. This chapter also contains a great part of our professional experience and the most frequently ratified as the existing solutions in this field of study. Although this book revises mycotoxicological concepts of the commercial swine industry, it also deals with some historical aspects, mold that produce mycotoxins, physical and chemical characteristics of mycotoxins, toxicity, clinical signs, lesions, diagnostics, and ways to temper the toxic effects of each mycotoxin.

5 This project was also possible thanks to the help of all those who contributed to the creation of LAMIC, institution that foments and supports the development of this science. We dedicate this book to our graduate students, researchers, and collaborators. We would like to thank Special Nutrients for making this book available in English and thus available to a global audience. Finally, we would like to thank our families for their unconditional support and dedication to this cause.

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MYCOTOXINS

7 MYCOTOXINS Mycotoxins are toxic substances that are the result of secondary metabolism of diverse strains of filamentous fungi. They are organic compounds of low molecular weight and low immunogenic capacity. They are present everywhere, nevertheless they are predominate in tropical and subtropical climates where fungal development is favored by the environmental conditions. The main fungal species that produce mycotoxins belong to the genera Aspergillus, Penicillium, Fusarium, Claviceps, Alternaria, Pithomyces, Mirothecium, Stachibotrys, and Phoma. In tropical and subtropical climates, like that of Brazil, fungal development is favored by factors such as optimal humidity and temperature conditions. More than five hundred mycotoxins produced by approximately one-hundred fungi are known. The most important can be divided into three groups: aflatoxins produced by fungi of the genus Aspergillus, such as A. flavus and parasiticus, ochratoxin produced by fungi of the genera Aspergillus and Penicillum, and fusariotoxins which are represented by trichothecenes, zearalenone, and fumonisins, produced by different species of the genus Fusarium (Table 1). Table 1 – Main mycotoxins, primary fungi producers, food supply prone to contamination, and favorable conditions for occurrence. Main fungi producers Food supply prone to contamination Main factors that lead to the production of mycotoxins

Mycotoxin Aflatoxins

Aspergillus Almonds, chestnuts, flavus and A. acorn, corn and grains parasiticus in general. Fusarium Corn and winter grains. Corn and winter grains. Corn and winter grains. Corn and stored grains.

Inadequate storage conditions.

Zearalenone

Low temperature associated with high levels of humidity. Dry season followed by high humidity and moderate temperature. Low temperature, high humidity, and storage problems. Storage deficiencies.

Fumonisins

Fusarium

Trichothecenes Ochratoxin A

Fusarium Aspergillus Penicillium

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When mycotoxins are ingested, by humans or animals, they can produce different deleterious health effects, resulting in negative economical, sanitary and commercial consequences. Besides other less toxic effects, ingesting mycotoxins can also negatively affect reproductive health because of their anabolic, estrogenic, carcinogenic, mutagenic, and teratogenic properties. The danger of mycotoxins is not just the possibility of lethal effects, but rather, the harm they present to different organs and systems, decreasing optimal productive performance. The first incidents of mycotoxicosis are present in the Old Testament, as the tenth plagues of Egypt, which is narrated in the books of Exodus and Job in the passage where Moses tries to free the Hebrews from Pharaoh’s control. There is evidence of the presence of mycotoxins in the plagues that demised flocks and herds, and caused tumors and ulcers in Egyptian animal and human populations. In the historical evolution of mycotoxins, the episode termed “Saint Anthony’s fire” which occurred in the Middle Ages, specifically during the XI and XVI century, is noteworthy because it affected the population of various European countries, particularly France. The disease was characterized by outbreaks of gangrene in the population which had consumed grains contaminated by sclerotia (rye ear) of the fungus Claviceps purpurea. The disease is a result of the vasoconstrictor properties of ergotamine, which obstructs peripheral circulation. Furthermore, the disease also promotes oxytocia and stimulates the central nervous system, followed by a noticeable depression. During the period between 1930 and 1940, stachybotryotoxicosis caused the deaths of tens of thousands of horses in the former USSR. This mycotoxicosis is caused by a toxin produced by fungi of the Stachybotrys genus, such as S. chartarum and S. alternans, which grow well in humid hay and straw. The clinical manifestations of this mycotoxicosis include neurological alterations caused by tremors, diminished visual capability, often occurring epidermal necrosis, leukopenia and ulcerations in the gastrointestinal tract. In the beginning of the XX century, specifically in the years from 1941 to 1945, Alimentary Toxic Aleukia (ATA diseases) was responsible for making a large number of Europeans sick, killing more than 100 thousand Russians. This was a result of ingesting trichothecenes produced by cryophilic fungi of the Fusarium genus, specially F. esporotrichioides, graminearum, and moniliforme. The disease has four stages of evolution, in this order: lesions in the upper digestive tract, hematopoietic damage, damage to the

9 nervous system, and damage to the endocrine system. In the XIX and XX century, an epidemic called yellow rice disease struck Japan and caused a great number of deaths due to consumption of moldy rice. The disease was attributed to citreoviridin, a cardiotoxic toxin (cardiac beriberi) caused by fungi of the Penicillium genus. Balkan nephropathy, a disease which affected eastern European countries from 1957 to 1958, was a result of consuming food sources contaminated by ochratoxin A, produced by fungi such as Aspergillus ochraceus and Penicillium viridicatum. Mycotoxins began receiving mayor scientific attention in the 1960s, when aflatoxins were found responsible for the deaths of more than one hundred thousand poultry in Europe. Advances in research demonstrated that mycotoxins contain extremely toxic properties which affect all mammals; therefore, mycotoxins have gradually gain scientific importance around the world. So much so that in 1988 fumonisins were attributed with different illnesses in domestic animals and were related to a large quantity of esophageal cancer outbreaks in humans. Today, more than five hundred chemical compounds are categorized as mycotoxins. Currently, knowledge concerning the physiology, production of toxins and development of fungi producers of mycotoxins are still limited. Nevertheless, it is known that mold grows in grains and cereals, especially in peanuts, corn, wheat, barley, sorghum, and rice because they contain nutritious substrates for fungal development. Cereals and grains can lose important portions of their nutritional value when contaminated with mycotoxins which can remain present for various years, even when fungi are no longer present. Fungal growth and mycotoxin production in cereals can occur in different phases of development: maturation, harvest, transportation, processing, or storage of grains. The difficulty of harvesting cereals in the correct stage of maturation and humidity could be another critical point that concerns the formation of mycotoxins. It is normal that cereals harvested with a high level of humidity facilitate fungi development before these cereals are dried, and even in storage, especially when these grains go through a drying process. Cereals harvested after the physiological maturation of the grain are subjected to high levels of humidity and to the onset of plagues that create favorable conditions for mold development. The transportation of cereals with high levels of humidity for long distances or prolonged periods of time, also favors the development of fungi, due to the formation of an adequate microclimate for such growth. Therefore, reducing cereal humidity through a drying process

10 is critical. The drying process, when done improperly, can also contribute to the proliferation of fungi in grains. In Brazil, the long lines and waiting time of trucks that transport cereal to the entrance of the drying facility frequently contribute to a hasty drying process resulting in deficiencies such as increased drier temperatures, producing mechanical and thermal damages to the grain, and consequently decreasing their immunity. Other factors that encourage fungal development and production of mycotoxins include a series of storage deficiencies, such as high levels of humidity, uneven distribution of cereal inside silos, variations in ventilation, and incidence of pests. Understanding the various factors that can increase the formation of these toxic metabolites is fundamental in order to diminishing the production of mycotoxins (Figure 1). Figure 1 – Intrinsic and extrinsic factors related to the production of mycotoxins.

Water Temperature Insects Time Fungi Load Substrate composition Oxygen Mechanical damages Fungi competition Fungi genetic Fungi interaction

Fungi growth and mycotoxins production

The different effects of mycotoxins are due to their specific chemical structures, varying reactions to their ingestion by different animals influenced by species, race, sex and age, environmental factors, nutritional status, and the presence of other chemical substances. (Table 2)

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Table 2 – Major mycotoxins, species affected, and main clinical signs and lesions. Mycotoxins
Aflatoxins Zearalenone Fumonisins Trichothecenes Ochratoxin A

Species most affected
All Swine Horses and Swine Monogastrics Swine and humans

Main clinical signs and lesions
Diminished weight gain, digestive disorders, hepatopathies, anorexia, ataxia, tremors and death. Hyperestrogenism syndrome (vulvovaginitis). Horse leukoencephalomalacia. Swine pulmonary edema. Feed reduction or refusal, digestive disorders with ulcerations and vomiting, and visceral hemorrhages. Nephropathy.

In spite of a series of variations, a susceptibility to mycotoxicosis can be estimated. In order to do this, it is necessary to quantify the level of feed contamination and feed consumption, which indicates the type and severity of each disease. Mycotoxicosis can be divided into acute and chronic disorders. The acute manifestations occur when individuals consume moderate to high doses of mycotoxins. Clinical syndromes and specific pathological signs can become present, depending on the mycotoxin ingested, the susceptibility of the species, the individual conditions of the organism, and the interaction or lack of interaction with other factors. Lesions depend on each mycotoxin (Table 3); however, the most commonly found are signs of hepatitis, hemorrhaging, nephritis, necrosis of the digestive mucosa or death. Chronic mycotoxicosis occurs when moderate to low doses are consumed. In these cases, the acute manifestations of intoxication are not present; nevertheless, characteristic clinical symptoms such as decrease in reproductive efficiency, feed conversion, growth rate, and weight gain are present. These characteristics are only detected by detailed observation and training, or by monitoring mycotoxins levels in feed. The clinical signs could be confused with other illnesses proceeding from mycotoxicosis or from nutritional deficiencies. There are few statistics available related to the incidence of mycotoxicosis, nevertheless there is a

12 general awareness that the “hidden danger” (chronic intoxication) is responsible for a large portion of the losses experienced in productive animals. The results of laboratory evaluations conducted in the last 17 years indicate that aflatoxins are the most prevalent mycotoxins in Brazil. As shown in Table 4, ochratoxin A and T-2 toxin contamination is not significant, in spite of the fact that the latter does not have a history of being diagnosed with adequate methodologies. One important effect caused by chronic mycotoxicosis is immunosuppression, leaving the individual predisposed to other illnesses whose pathogens easily multiply with the decrease of the animal’s resistance to illness. In addition, ingesting feed contaminated with mycotoxins causes inadequate responses to vaccinations.

13 Table 3 - Effects of main mycotoxins in swine.
Mycotoxin Swine stage Level in feed (?g/kg) 10-100 200-400 Growing / finishing 400-800 800-1,200 1,200-2,000 Sow/piglet Prepubescent gilts Zearalenone (F-2) Gestating Lactating sows Replacing gilts 500-750 1,000-3,000 3,000-10,000 25,000 25,000-50,000 >25,000 1,000-20,000 Fumonisins All swine >20,000 2,000-8,000 8,000-10,000 < 2,000 T-2 Toxin Growing/ finishing 8,000 16,000 2,000 Deoxynivalenol (DON or vomitoxin) Growing/ finishing 5,000-10,000 12,000 20,000 200 Growing/ finishing Females/ piglets 1,000 4,000 3,000-9,000 Principal clinical signs

Loss of productivity, without visible clinical signs. Poor growth and feed efficiency. Hepatopathies (friable and yellow-bronze liver); immunosuppression. Significant decrease in feed intake and growth, icterus and hypoproteinemia. Icterus, coagulopathy, anorexia, and deaths. Reproductive disorders, weak piglets due to contamination through milk. Edematose vulva, reddened and prolapsed rectum. Edematose vulva, retention of corpus luteum and anestrus. Repeated heat. Small litter, weak piglets, edematose and reddened vulvas in neonatals. Pseudogestation, nymphomania, and persistent infertility. Hepatopathies, tumors, and decrease of productivity. Enlarged heart. Sharp pulmonary edema, hepatopathies, and decrease in feed intake. Decrease in feed intake and ADG, epidermic and oral irritation, and intestinal epithelium hypertrophy. Complete refusal of feed. Hemorrhaging and enteritis. Decrease in feed intake. Complete refusal of feed. Decrease in feed intake and growth. Decrease in feed intake, and weight loss. Complete refusal of feed. Vomiting. Renal lesions seen at processing. Polyuria, uremia, decrease of ADG. Severe renal failure. No alterations in estrous cycle or conception rates.

Aflatoxins

Diacetoxyscirpenol (DAS)

Growing/ finishing

Ochratoxin A

14 Table 4 – Results from analysis of mycotoxins in different feeds, taken during the period of 1994 to 2010 by the Laboratory of Mycotoxicological Analysis, LAMIC/UFSM. Toxin Aflatoxins1 Zearalenone Fumonisins2 DON Ochratoxin A T-2 Toxin Total
1 2

# Samples Analyzed 138,875 105,509 45,558 39,451 30,973 16,939 377,305

Positive % 35.8 29.8 64.0 40.1 4.0 1.0 28.4

Average (?g/kg)

Maximum (?g/kg) 16,861.5 17,000.0 289,283.0 23,740.2 404.3 2,133.0

8.4 74.1 1591.0 313.1 0.6 1.2

Results from the sum of aflatoxins B1+B2+G1+G2 Results from the sum of fumonisins B1+B2

With advances in research, mycotoxins have become scientifically significant at a global level. Among the hundreds of known mycotoxins, aflatoxins are considered the most important. They are produced by the fungus genus Aspergillus and are classified as B1, B2, G1 and G2. These fungi develop in grains, above all in peanuts and corn. When its toxins are ingested the immediate reaction is a decrease in productivity, as well as the carcinogenic, mutagenic, teratogenic, heptatoxicity and immunosuppression effects. Aflatoxin B1 is the most toxic of the group, mainly affecting swine and poultry. Aflatoxins were categorized as Class 1 human carcinogens by the International Agency for Research on Cancer (IARC) (IARC, 1987). The Brazilian Ministry of Agriculture established a maximum limit of 20 ?g/kg, comprising B1, B2, G1 and G2 in food sources destined for human consumption (Brazil, 1996). The maximum limit recommended for aflatoxins in feed by the Brazilian Ministry of Agriculture is 50 ?g/kg (Brazil, 1988). The ingestion of aflatoxins decreases meat, milk, and egg production, and they can be carried by some of these food sources. Therefore, aflatoxins and products of their biotransformation can be isolated in these food sources and their derivatives, such as processed meats, cheeses, and yogurts.

15 Zearalenone is a toxin produced by various species of the Fusarium genus, especially by Fusarium graminearum. It causes alteration in female swine reproductive system, resulting in reproductive complications. Ochratoxin A is produced by fungi such as Aspergillus ochraceus and alutaceus, and fungi of the Penicillium genus. The main effect of Ochratoxin A for swine is nephrotoxicity. Trichothecenes is a group comprised of chemically similar metabolic toxins, produced by various species of fungi such as: Fusarium, Cefalosporium, Myrothecium, Stachybotrys, and Trichoderma. Approximately 200 mycotoxins make up this group, T-2 Toxin, Diacetoxyscirpenol (DAS) and Deoxynivalenol (Vomitoxin or DON) being the most well known. These toxins inhibit protein synthesis and interfere with DNA and RNA synthesis. Besides lesions in the digestive tract, especially on the mouth, pharynx, and esophagus, which are important for clinical diagnosis; animals show a decrease of productivity, as well as a deficiency in their immune system. The chemical structures of fumonisins began to be discovered in 1988. Fumonisins are produced by fungi of the Fusarium and Alternaria genus. These fungi are present throughout the world. The most prevalent fumonisin is fumonisin B1, responsible for pathological effects such as leukoencephalomalacia in horses, which is characterized by liquifactive necrosis of the white substance of the central nervous system, pulmonary edema in swine, hepatopathy in various species, and esophageal cancer in humans. The incidence of leukoencephalomalacia in southern Brazil is seasonal, with the majority of cases being observed during the coldest months when animal feed is supplemented with grain based rations. The corn used as a dietary supplement is frequently responsible for the presence of said pathology. It is known that Fusarium moniliforme develops in corn containing high humidity in favorable temperatures. The growth of the fungus is accompanied by the development of fumonisins in feed ingredients, which produces pathologies in animals. Preventive measures consist of appropriate conservation of grains accompanied by laboratory tests to detect fumonisins. Presumable diagnosis of mycotoxins is based on clinical signs observed in intoxicated animals, and analysis of environmental data concerning harvesting and storing conditions of grains used in feed for swine. Normally the introduction of a new batch of feed,

16 sometimes containing altered physical characteristics is associated with intoxication. A more reliable diagnosis is done by analyzing the presence of mycotoxins in the feed of the affected animals. The most commonly used techniques are ELISA kits analyses, Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). Mass spectrometry (GC/MS, LC/MS and LC/MS/MS) as a method of diagnosis is a recent development. It has been proven that these are the most efficient methods, and they will surely substitute all others in the near future. Figure 2 – Equipment used to detect and quantify mycotoxins. (A) High Performance Liquid Chromatography. (B) High Performance Liquid Chromatography coupled with Mass/Mass spectrometry.

The treatment of mycotoxins represents one of the greatest challenges to clinical veterinary. The removal of contaminated feed is the first measure that should be adopted, and seems to improve prognosis. Even though there is a lack of scientific evidence, the inclusion of additional levels of sulfur amino acid to the feed has been used to decrease mycotoxicosis. The main prophylactic measures consist of adopting harvesting and handling techniques that make fungal growth difficult, such as the harvesting of grains immediately after reaching their physiological maturity, making them less exposed to weather, and drying and stowage in adequate granary for each type of grain or grain product. Knowledge regarding fungi physiology and development and production of toxins are still insufficient, precisely because research regarding production care and storage for the majority of grains produced is limited. The choice of a given corn strain that is resistant to fungal development can diminish the probability of the presence of mycotoxins. For example, varieties of corn

17 that possess high concentrations of linoleic acid can be more resistant to fungal development of the Aspergillus genus. Hence, monitoring grains and their byproducts through adequate sampling techniques and mycotoxicological analysis before grains are used are essential practices, especially when given grains have been exposed to environmental conditions that are favorable for fungi growth. The use of organic acids can help conserve feed in risky situations. The use of additives, whether natural or modified by the addition of enzyme compounds or biological compounds to the feed, deserves scientific study. These additives have shown promising results for swine in some field cases, which are still not entirely clear. BIBLIOGRAPHY AZEV?DO, I. G.; GAMBALE, W.; CORR?A, B. Mycoflora and aflatoxigenic species of Aspergillus spp. isolated from stored maize. Rev. Microbiol., v. 25, n. 1, p. 40-50, 1994.
BRASIL – Leis e decretos. Ministério da Agricultura. Portaria n. 7, de 9 de novembro de 1988. Diário Oficial da Uni?o, de 9 de novembro, Se??o I, página 21.968, Brasília, 1988. BRASIL – Leis e decretos. Ministério da Agricultura. Portaria n. 183, de 21 de mar?o de 1996. Diário Oficial da Uni?o, de 25 de mar?o, Se??o I, página 4929, Brasília, 1996. BRASIL – Leis e decretos. Ministério da Saúde. Resolu??o n. 34/76. Diário Oficial da Uni?o, de 19 de janeiro, Se??o I, página 710, Brasília, 1977. CAST, Council for Agricultural Science and Technology. Mycotoxins: Economic and Health Risks. Report 116, 1989. CLEVSTROM, G. Studies of fungi flora of plants and feeds and the influence of formic acid on growth and aflatoxin production in Aspergillus flavus. Upsala: Sweden, 1986. DILKIN, P.; MALLMANN, C.A.; ALMEIDA, C.C.A.; CORR?A, B. Robotic automated clean-up for detection of fumonisins B1 and B2 in corn and corn-based feed by high-performance liquid chromatography. J. Chromatogr. A, v. 925, n. 1-2, p. 151-7, 2001. EDDS, G.T. Acute aflatoxicosis: a review. J. Am. Vet. Med. Assoc., v. 162, n. 4, p. 304-9, 1973. HARVEY, R.B.; KUBENA, L.F.; PHILLIPS, T.D.; HUFF, W.E.; CORRIER, D.E. Prevention of aflatoxicosis by addition of hydrated sodium calcium aluminosilicate to diets of growing barrow. Am. J. Vet. Res., v. 50, n. 3, p. 416-20, 1989. IARC (INTERNATIONAL AGENCY FOR RESEARCH ON CANCER). Overall evaluations of carcinogenicity: AN UPDATING OF IARC MONOGRAPHS ON THE EVALUATION OF CARCINOGENIC RISK TO HUMANS. Lyon: WHO, 1987. v. 1-2, supl. 7, p. 83-7. IARC (INTERNATIONAL AGENCY FOR RESEARCH ON CANCER). Toxins derived from Fusarium moniliforme: fumonisin B1 and B2 and fusarin C. In: IARC monographs on the

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evaluation of carcinogenic risks to humans: some naturally occurring substances, food items and constituents, heterocyclic aromatic amines and mycotoxins. 56, 1993 Lyon. Lyon: IARC: 1993. p. 445-66.

MALLMANN, C.A. Epidemiologische Studien zum vorkommen von Ochratoxin A im Serum von
Schweinen auf der basis von Schlachthof- Und Bestandsuntersuchungen. Tesis, T ier?rztliche

Hochschule Hannover, 1993. 152p.
MALLMANN, C.A.; DILKIN, P. Micotoxinas: Clínica e Diagnóstico em Suínos. In: CORR?A, M. N.; LUCIA, T. JR.; DESCHAMPS, J. C. Tópicos em Suinocultura. Pelotas, 2003, v. 1, p. 191-222. MALLMANN, C.A.; SANTURIO, J.M.; ALMEIDA, C.A.A.; DILKIN, P. Fumonisin B1 in cereals and feeds from southern Brazil. Arq. Inst. Biol., v. 68, n. 1, p. 41-5, 2001. MALLMANN, C.A.; SANTURIO, J.M.; DILKIN, P. Equine leukoencephalomalacia associated with ingestion of corn contaminated with fumonisin B1. Rev. Microbiol., v. 30, p. 249-52, 1999. MEIRELES, M.C.A.; CORR?A, B.; FISCHMAN, O.; GAMBALE, W.; PAULA, C.R.; CHACONRECHE, N.O.; POZZI, C.R. Mycoflora of the toxic feeds associated with equine leukoencephalomalacia (ELEM) outbreaks in Brazil. Mycopathologia, v. 127, n. 3, p. 183-8, 1994. MILLER, D.M.; CROWELL, W.A.; STUART, B.P. Acute aflatoxicosis in swine: Clinical pathology, histopathology, and electron microscopy. Am. J. Vet. Res., v. 43, n. 2, p. 273-7, 1982. NEAL, G.E. Participation of animal biotransformation in mycotoxin toxicity. Revue Méd. Vét, v 149, p. 555-560, 1998. NEWBERNE, P.M. Chronic aflatoxicosis. J. Am. Vet. Med. Assoc., v. 163, n. 11, p. 1262-7, 1973. NEWBERNE, P.M.; BUTLER, W.H. Acute and chronic effects of aflatoxin on the liver of domestic and laboratory animals: A review. Cancer Res, v. 29, n. 1, p. 236-50, 1969. OMS (ORGANIZACI?N MUNDIAL DE LA SALUD). Critérios de salud ambiental 11: Micotoxinas. México: OMS, 1983. 131 p. PIER, A.C. An overview of the mycotoxicosis of domestic animals. J. Am. Vet. Med. Assoc., v. 163, p. 1259-61, 1973.

RILEY, R.T.; WILLIAM, P.; NORRED, P. Fungal toxins in foods: recent concerns. Annu. Rev. Nutr., v. 13, p. 167-89, 1993.
RODRIGUEZ-AMAYA, D. B.; SABINO, M. Mycotoxin research in Brazil: the last decade in review. Braz. J. Microbiol., v. 33, n. 1, p. 1-11, 2002. SCHOENTAL, R. A corner of history: Moses and Mycotoxins. Prev. Med., v. 9, n. 1, p. 159-61, 1980. SCHOENTAL, R. Mycotoxins and the Bible. Perspect. Biol. Med., v. 28, n. 1, p. 117-20, 1984. SILVA, J.B.; POZZI, C.R.; MALLOZZI, M.A.B.; ORTEGA, E.M.; CORR?A, B. Mycoflora and occurrence of aflatoxin B1 and fumonisin B1 during storage of Brazilian sorghum. J. Agric. Food Chem., v. 48, n. 9, p. 4352-6, 2000.

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WILSON, D. M.; SANGSTER, L. T.; BEDELL, M. B. Recognizing the signs of porcine aflatoxicosis. Vet. Med. v. 7, p. 974-6, 1984. WORLD HEALTH ORGANIZATION. Mycotoxins Environmental Health Criteria, 11, Genebra, WHO, 1979, p. 21-84. www.lamic.ufsm.br

ZERINGUE, H.J.; BROWN, R.L.; NEUCERE, J. N. Relationships between C6-C12 alkanal and alkenal volatile contents and resistance of maize genotypes to Aspergillus flavus and aflatoxin production. J. Agric. Food Chem., v. 44, p. 403-7, 1996.

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AFLATOXINS

21 AFLATOXINS Summary Aflatoxins are secondary metabolites of the toxigenic fungi of the Aspergillus genus, specifically A. flavus and A. parasiticus. Among the hundreds of known mycotoxins, aflatoxins are considered the most important in Brazil. Various compounds have been described as aflatoxins, nevertheless, only aflatoxins B1, B2, G1 and G2 have been identified as natural contaminants of grains and feed. Generally, aflatoxin B1 is the most prevalent in various agricultural products, and it is also the most toxic amongst these aflatoxins. Aflatoxins were initially known to cause “Turkey X Disease”, whose toxicosis cause high death rates for these animals in England in 1960. After this intoxication, aflatoxins became recognized as highly hepatocarcinogenic, mutagenic, and teratogenic substances for animals and humans. They can cause various toxic effects in animals, predominantly affecting the liver, thus compromising the health and productivity of intoxicated animals. Swine are particularly susceptible to intoxication due to aflatoxins. Acute intoxications frequently lead to the death of animals, nevertheless, the greatest loss results from subacute and chronic intoxications. In these circumstances, animals ingest less feed and there is a decline in productivity, a decrease of immunological capabilities, and various pathological disorders in various organs. These symptoms result in the general sickness of the affected animals. History Mycotoxins received significant scientific importance due to the high number of mortalities caused by aflatoxin intoxication of animals. In England, in 1960 an apparently new illness known as Turkey X Disease was recorded; more than one hundred thousand turkeys, twenty thousand ducks, and hundreds of poultry died after ingesting peanut meal allegedly imported from Brazil, and contaminated with aflatoxins. Aflatoxicosis outbreaks were immediately observed in other animal species, such as bovine, swine, sheep, and chicken that had ingested the peanut meal of the same origin. At the same time, peanut meal imported from other countries produced the same toxicological symptoms when it was ingested by animals.

22 The principal clinical signs present in the intoxicated animals were a loss of appetite, lethargy, weakness, and death. Histopathological examinations revealed necrotic lesions in the liver, as well as disseminated hemorrhages. After a series of investigations, the intoxications were correlated to a toxin produced by the fungi of the Aspergillus genus, especially A. flavus. Researchers had a difficult time determining that the toxin was produced by that fungal species, because the fungi were absent in most of the samples tested. This absence is due to the fact that even after the death and decomposition of fungi, the toxins they produced persist in feed and ingredients. One of the first great discoveries related to toxins in peanut meal was the existence of a toxic substance that could be isolated by using high polarity organic solvents. This isolated extract emits fluorescence under the incidence of ultraviolet light; and its toxicity was proven by reproducing toxicological clinical symptoms by administrating the purified extract to healthy animals. Proof of fluorescence emission by the toxins, when submitted to the incidence of ultraviolet light, was a major scientific discovery because this characteristic was of vital importance for the development of analytical methodologies, such as Thin Layer Chromatography, to identify and quantify the newly discovered toxins. In 1962 these toxins were named aflatoxins because they were produced by fungi of the species A. flavus. Initially, only two toxins were identified through Thin Layer Chromatography. They were named aflatoxin B and aflatoxin G, consistent with the emission of blue or green light under the incidence of ultraviolet light. Soon afterwards, two other toxins emitting blue light were discovered. These were named aflatoxins B1 and B2. Two other substances emitting green light were also discovered; and named aflatoxins G1 and G2. Research following the discovery, identification, and characterization of aflatoxins indicate that there are great differences in susceptibility, especially related to the species, age and sex of the animal. In the last three decades, aflatoxins and their toxicosis were widely studied in different animal species, as well as in humans. Aflatoxins constitute one of the most potent natural hepatocarcinogens known, responsible for various outbreaks of hepatocellular carcinoma in a variety of animal species. Aflatoxin B1 is considered the most potent natural carcinogen known.

23 Etiology Aflatoxins are secondary fungal metabolites, and form part of the bifuranocumarin group. They are formed by heterocyclic molecules, with oxygen atoms and furano rings that differ amongst each other by small variations in their basic molecular structures. They are produced by Aspergillus flavus and A. parasiticus. Strains of A. nomius and A. pseudotamarii produce these mycotoxins in smaller quantities. More than twenty substances of aflatoxins are known, nevertheless, the most common in feed are aflatoxins B1, B2, G1, and G2 (Figure 3). In the majority of isolated fungi, aflatoxin B1 is the most frequently produced, along with B2, which is produced in large quantities by A. flavus; whereas A. parasiticus produces all four in similar quantities. Figure 3 – Chemical structure of the four naturally occurring aflatoxins.

O O

O O

O

O

O

O A fla to x in B Aflatoxin Ba 11 O O

OCH3

O

O A fla to x in a2 B Aflatoxin B2

OCH3

O O O

O

O O

O

O
Aflatoxin G A fla to x in a 11G

OCH3

O

O A fla to x in Aflatoxin G2a2G

OCH3

Other aflatoxins have already been isolated from animals’ milk, meat, and urine, as is the case of aflatoxins M1 and M2, which are metabolites of aflatoxins B1 and B2, respectively. These mycotoxins are produced in the liver few hours after consuming contaminated feed.

24 Aflatoxins are starkly fluorescent to ultraviolet light (365 nm). Toxins B1 and B2 emit a fluorescent blue, hence the letter B. Aflatoxins G1 and G2, under the incidence of the same light wave, emit a green light, therefore deriving its letter from the word green. They are fairly soluble in moderately polar solvents such as chloroform, methanol, and acetonitrile. They have a low solubility in water and a melting point close to 269°C (Table 5). They are particularly sensitive to ultraviolet light, especially when they are dissolved in polar solutions. They are also destroyed by autoclave in the presence of ammonia and when treated with hypochlorite. Table 5 – Physical-chemical characteristics of aflatoxins. Aflatoxin Chemical formula B1 B2 G1 G2 M1 M2 Aflatoxicol C17H12O6 C17H14O6 C17H12O7 C17H14O7 C17H12O7 C17H14O7 C17H14O6 Molecular mass 312 314 328 330 328 330 314 Melting point (°C) 269 286-289 224-246 237-240 299 293 230-234 Fluorescence emission (nm) and color 425 – blue 425 – blue 450 – green 450 – green 425 – violet-blue violet 425

Presence of aflatoxins in feed The presence and magnitude of feed contamination with aflatoxins varies due to geographical and seasonal factors, as well as conditions in which agricultural products are cultivated, harvested, and stored. Crops produced in tropical and subtropical regions are more prone to contamination than are those in temperate regions, precisely because the optimal conditions for the production of toxins are predominant in regions of high Aflatoxin are principally produced with Aw >0.7, high air humidity, and temperatures between 24 and 35?C.

humidity. Toxigenic fungi can invade and develop in a wide variety of substrates such as

25 cereals, seeds, and food sources during periods of growth, cultivation, harvest, transport, processing, and storage. The presence of fungi, including those species which produce aflatoxins, does not necessarily signify a presence of toxins. Similarly, the presence of these toxins is not necessarily related to the presence of a producing fungus, since the toxins present great stability in grains, even after the deterioration of the producing fungus. The most important factors for the growth of toxigenic fungi of the Aspergillus genus and the production of aflatoxins in stored cereals are high relative air and substrate humidity, and the temperature at which they are stored. Relative air humidity of 80 to 85% with water activity (Aw) superior to 0.7 in cereals and temperatures between 24 and 35°C represent favorable condition for the production of aflatoxins. According to OMS (1983), the minimum, optimal, and maximum temperatures for the production of aflatoxins are 12, 27 and 40-42°C, respectively. Therefore, the contamination of agricultural products and feed by aflatoxins has a global scope. Its levels of occurrence are influenced above all by temperature, humidity, and substrate type. The interaction between these and other different factors will determine the amount of toxins produced. The concentrations detected during various research and monitoring procedures indicate that levels showed significant variations in different regions. Although significant variations between different years in a determined region can be observed, these are probably influenced by climatic conditions, as was the case in the evaluations done in Brazil by LAMIC/UFSM over the last 17 years (Table 6). According to studies done by the Council for Agricultural Science and Technology in 1989, approximately 25% of cereals harvested around the world are contaminated by mycotoxins. Corn, prominently used in feed, has an important role in contamination, for humans as well as for animals. In Brazil, various studies were done in order to detect the presence of aflatoxins in feed. The toxin was widely detected and concentrations found vary significantly. Research carried out in S?o Paulo, Brazil in 1988 detected the presence of aflatoxins in 54% of peanut samples and 3.6% of corn samples. In other observations, in which hundreds of samples of corn from different regions of Brazil were analyzed, 12.3% presented different concentrations of aflatoxins.

26 Table 6 – Number of samples, percentage of positivity and average contamination by total aflatoxins in the last 17 years of routine analysis by LAMIC/UFSM.
Year Number of samples Average of positive samples (%) 57.3 40.9 35.0 33.2 21.4 40.6 31.3 45.3 62.2 48.3 31.4 36.0 41.5 37.2 27.5 31.0 23.2 37.84 Contamination average (?g/kg) 50.9 20.8 33.9 13.3 28.8 8.8 9.5 14.6 11.1 11.2 4.8 3.9 12.2 5.4 3.1 7.0 8.3 14.56 Percentage of contamination >10 (?g/kg) 27.9 18.5 18.7 11.0 9.4 11.2 11.3 11.1 15.5 13.6 4.8 5.4 12.8 7.4 5.4 7.7 3.3 11.47

1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 Total

763 785 1713 3051 3536 4376 5535 6199 7769 8341 8682 10792 14798 16848 16507 15304 13867 138,875

Three hundred eighty-two samples of corn used in animal feed were studied in 1992 in Southern Brazil. Aflatoxins were detected in 28.5% of these samples, with an average concentration of 1.9 ?g/kg. After one year, 1,131 samples of grains and feed were analyzed, of which 44.3% were positive for mycotoxins, 89.9% of these were aflatoxins. In 1997 the prevalence of mycotoxins was studied in 5,335 samples of cereals and byproducts from the Southern Brazil. The authors found that 42.6% of the analyzed samples were positive for mycotoxins, with an average level of 34.5 ?g/kg. The main feed ingredient analyzed was corn, 2,460 samples, which presented a positivity of 51.8%. Evaluations carried out by LAMIC/UFSM, during the last 17 years demonstrate that the occurrence of aflatoxins in corn presents a frequency of positivity of 47.2% in 63,907 samples analyzed routinely in said laboratory; with a contamination average of 10.0 ?g/kg. The general positivity of 138,875

27 samples of all feed ingredients which were analyzed was 35.9%, with a contamination average of 8.3 ?g/kg; slightly inferior than in corn because of lower averages of aflatoxin concentrations in other feed ingredients. The simultaneous occurrence of aflatoxins with other mycotoxins was found in various agricultural products and feed ingredients. This is of significant importance because the toxic effects of these mycotoxins can be synergistic with those of aflatoxins. Although a large portion of the research The co-occurrence of aflatoxin with other mycotoxins must always be considered.

reveals that the co-occurrence of aflatoxins with fumonisins, other mycotoxins such as trichothecenes, zearalenone and ochratoxin A, have been isolated in feed containing aflatoxins. In other evaluations conducted by LAMIC/UFSM, the co-occurrence of mycotoxins was confirmed. Table 7 illustrates the co-occurrence of aflatoxins with other important mycotoxins. Table 7 – Percentage of co-occurrence and concentrations of aflatoxins, zearalenone, fumonisins and deoxynivalenol in the last 17 years (1994 to 2010) in samples of cereals and feed routinely analyzed by LAMIC/UFSM.
Mycotoxins Afla1 + Zea2 Afla + FBs3 Afla + Don4 Afla + Zea + FBs Afla+ Zea + Don
1 2 3 4

Number of samples 93,358 40,802 23,869 30,427 21,262

Co-occurrence (%) 9.2 24.3 8.3 10.4 4.0

Average 1* (?g/kg) 7.4 7.3 3.0 7.3 3.7

Average 2* (?g/kg) 59.2 2084.8 109.0 111.0 160.3

Average 3* (?g/kg) ------2686.0 129.1

Aflatoxins B1 + B2 + G1 + G2. Zearalenone. Fumonisins B1 + B2. Deoxynivalenol.

* Averages 1, 2, and 3 refer to the concentrations of mycotoxins that present both toxins, and is in accordance with the order cited in the column designated Mycotoxins.

28 The Brazilian Ministry of Health and The Ministry of Agriculture, Husbandry, and Supply established the maximum limit permitted of aflatoxin in food destined for human consumption at 20 ?g/kg, comprising aflatoxins B1, B2, G1 and G2. The maximum limit of aflatoxins in feed accepted by the Brazilian Ministry of Agriculture, Husbandry, and Supply is presently set at 50 ?g/kg. Animals intoxicated with aflatoxins normally store and eliminate the products of their biotransformation. Therefore, aflatoxins and products of their biotransformation can be isolated in these foods and derivatives such as meat, milk, cold meats, cheeses, and yogurts. Toxicity of aflatoxins Of the four main mycotoxins of the group, aflatoxin B1 is the most toxic; the liver being its principal target organ. When compared to the magnitude of the toxicity of aflatoxin B1, that of aflatoxins G2, B2, and G1 are of 10, 20, and 50% respectively. Besides being hepatotoxic, aflatoxins are also highly mutagenic, carcinogenic, and possibly teratogenic in animals. Due to their high toxicity, aflatoxins are considered Class 1 human carcinogens by the International Agency for Research on Cancer (IARC). They present DL50 between 1 and 50 mg/kg for the majority of productive animal species, nevertheless, they are especially toxic for swine, presenting a DL50 of 0.62 mg/kg (Table 8). Aflatoxins M1 and M2 are products of aflatoxins’ B1 and B2 biotransformation, respectively. These products are especially important in suckling pigs because they are eliminated through the milk of intoxicated animals. The clinical manifestations of aflatoxicosis can be acute and chronic. Chronic illness occurs when moderate to high doses of mycotoxins are consumed, and clinical signs become evident within hours to a week after the initial consumption of the contaminated feed. In those cases, clinical signs and specific clinical pathological symptoms can appear, depending on the susceptibility of the animal species. The most frequent lesions are related to hepatitis, hemorrhage, nephritis and enteritis, followed by death. Chronic mycotoxicosis occurs when there is a consumption of moderate and low doses. In these circumstances, when animals present clinical toxicological symptoms, the signs are discrete and difficult to diagnose, and therefore can extend for weeks and months. Animals present clinical symptoms characterized by a reduction of reproductive efficiency, and deterioration of feed conversion, growth rate, and weight gain. These symptoms are detected through careful observations by experienced growers or by a permanent program of monitoring and analyzing mycotoxins in feed.

29 Clinical signs of chronic aflatoxicosis can be confused with a nutritional deficiency and/or other illnesses. Affected animals can also exhibit other illnesses as a result of decreased immunity. In most cases, young animals are the most sensitive to aflatoxicosis, and a difference in susceptibility between the sexes can be observed. Generally, females are more resistant than males. This has already been scientifically proven in rodents. The difference in susceptibility in animals of the same sex in a given lot is easily detected by the uneven growth of the animals, which tends to increase with the increase in doses or intoxication period. The lack of uniformity of the lots is also species. Table 8 – Values of lethal dose 50 (DL50) of aflatoxin B1 in different species. Species Rabbit Duckling Cat Swine Rainbow trout Canine Guinea Pig Sheep Monkey Chicken Mouse Hamster Rat (male) Rat (female) DL50 (mg/kg body weight) 0.3-0.5 0.34-0.56 0.55 0.62 0.81 (intraperitoneal) 1.0 1.4-2.0 2.0 2.2 6.5-16.5 9.0 10.2 7.2 17.9 Patterson, 1973 and Christensen et al. 1976. Aflatoxicosis causes lack of uniformity in intoxicated animals of the same lots.

severely affected by the large variety in individual susceptibility in animals of the same

30

Toxicokinetics of aflatoxin B1. Differences in susceptibility to the toxic effects of Aflatoxin B1 in diverse species stem from variations in absorption, distribution, biotransformation, and elimination of this toxin. The presence of specific enzymes responsible for the biotransformation is considered of great importance in the susceptibility of various species to the acute or chronic effects of aflatoxins, because toxicity is exercised by the products of its biotransformation. Absorption Aflatoxins are absorbed by passing through a membrane barrier from an external medium to the interior of the organism, reaching the blood stream. As a result of the lipid composition of the cells that make up membranes, the liposolubility of the xenobiotic absorbed is of fundamental importance because it facilitates the passage through this membrane barrier. Therefore, aflatoxins, because of their high liposolubility, can easily be absorbed by the skin, lungs, and gastrointestinal tract. According to the results of various researchers, the absorption of aflatoxins through cutaneous and respiratory channels is of great importance for professionals working in cereal mills. However, the easiest channel of absorption of aflatoxins occurs through the gastrointestinal tract, by ingesting contaminated feed. The absorption of aflatoxins is immediate after exposure, and seems to be completely absorbed when administered orally in combination with feed, especially when it is only moderately contaminated. Distribution The blood and lymph nodes distribute aflatoxins to different organs and tissues immediately after absorption. Most of the toxin is distributed to fat and soft tissue. Concentrations of aflatoxins occur in different organs, but the largest quantities can be detected in those organs responsible for biotransformation. Therefore, the liver and kidneys present the highest concentrations of aflatoxins and products of their biotransformation. Concentrations are usually inferior in other tissues and organs such as muscles, connective tissue, skin, pancreas, and lungs.

31

Biotransformation The biotransformation of aflatoxins occurs in the liver. The principal hepatic enzymes are located in the soft endoplasmatic reticulum (microsomes), and in the soluble material (cytoplasma). Reactions are promoted by catalyst enzymes whose reactions are divided into two phases. The first phase consists of oxidation, reduction, and hydrolysis, making molecules more hydrophilic. During the second phase, the compounds initially produced are combined with endogenous substances (sulfates, glutathione, amino acids, and methyl and acyl groups), with the objective of facilitating removal (Figures 4 and 5).

Figure 4 – Diagram of the biotransformation of aflatoxin B1 and its hydroxilated metabolites (AFM1, AFQ1 AFP1 and AFB2a), according to OMS 1983.

32 Figure 5 – Aflatoxin B1 and its biotransformation products, according to Leeson et al. (1995).
O AFLATOXICOL O OH

O O

O

AFLATOXIN B1 AFLATOXINA

1

O

O

OC H3

O

O O

OCH3 O

O AFLATOXINA AFLATOXINB B1 1 O 8,8, 9 EP?XIDO 9 EPOXIDE O O O O AFLATOXINA P1 AFLATOXIN P O
1

O AFLATOXINA AFLATOXIN Q1 O
1

H OH

OCH3 O

O

O O

OCH3 O

O AFLATOXINA B2a 2a AFLATOXIN B H

O

O O

OH O

OH

O

O

OCH3

AFLATOXINA AFLATOXINM M1 1 O OH

O

O

OCH3

The process consists of reversible and

irreversible

molecular

alterations.

Detoxification occurs by irreversible reactions whose biotransformation products are hydroxylated and hydrosoluble metabolites such as aflatoxins M1, Q1, P1 and B2a, which are generally less toxic that their precursors. Reversible detoxification consists in the formation of aflatoxicol, which will return to its original aflatoxin B1 form by means of oxidation reactions that occur in the microsomal dehydrogenase. The main activation reaction of aflatoxin B1 is characterized by the epoxidation of the molecule, forming 8,9-epoxide of aflatoxin which has covalent bonding properties with nucleic acids, diminishing protein production, as well as causing mutagenic, teratogenic, and carcinogenic reactions. These reactions occur in the enzymatic cytochromic system P450

33 which is formed by the enzymes responsible for the principal oxidation reactions, especially epoxidation of covalent bonds, important in the activation of xenobiotics. During the first phase of biotransformation, the enzymes of cytochrom P450 convert aflatoxin B1 into different hydrosoluble products. The capacity of detoxification determines, to a large extent, the susceptibility of each species to become intoxicated by aflatoxins, and this is a result of the presence, in larger or smaller concentrations of enzyme components of cytochrome P450, which can produce more or less toxic derivatives. Elimination The capacity to detoxify aflatoxins by different mammals varies greatly between species. Generally, it is significantly influenced by factors such as sex, age, health state, and nutritional status. The initiation of detoxification generally occurs in the liver, coinciding with the biotransformation of aflatoxin B1 into pharmacologically active byproducts. It is believed that this activation occurs in the nasal mucosa of swine, Aflatoxins can have a significant influence on respiratory diseases.

where higher quantities of products of biotransformation are usually found, instead of the liver. Thus, the concentration of aflatoxin B1 derivatives in hepatic tissue of swine and the occurrence of hepatocellular carcinoma is less than the incidence of upper respiratory carcinoma. The initiation, advancement, and aggravation of respiratory illnesses in swine could be significantly elevated because of the high concentration of bioactive forms of aflatoxin in the respiratory mucosa cells. The detoxification of aflatoxin B1-8,9-epoxide and aflatoxin M1 in mammal tissue is achieved by the combination of glutathione catalyzed by glutathione-s-transferase enzyme. However, it is estimated that about 0.5% of aflatoxin B1 that is ingested, will be eliminated through milk in the form of aflatoxin M1. The elimination of aflatoxins through eggs was found in various studies done with laying hens. Aflatoxin B1, or the products of its biotransformation (aflatoxin B2a, M1 and aflatoxicol) were distributed amongst the different parts of the eggs, reaching maximum concentrations 4-5 days following the initial administration of a constant dose of the toxin. Decline of the concentrations occurred during a similar period. Aflatoxins and the products of their biotransformation are principally eliminated through bile and feces, corresponding to approximately 60% of the ingested dose. The rest was eliminated, in similar proportions, through respiration, in the form of CO2, and urine.

34

Residues Aflatoxins can be quantified in the organs and tissues of different species such as bovines, swine, and poultry that have consumed feed contaminated with aflatoxins. The liver usually presents the highest concentrations. Aflatoxins B1, B2 and M1 are also frequently isolated in other organs and muscular tissues, such as meat consumed by humans. However, the levels normally found in those tissues are low, as can be seen in tables 9 and 10.

Table 9 – Aflatoxicol and aflatoxin B1 and M1 residues in tissues of piglets intoxicated with a single dose (1 mg/kg live weight) contained in rice culture (B1/G1 = 4.3/1) . Hours after intoxication 22 22 24 24 24 72 72 72 Tissue Kidney Liver Kidney Liver Muscle Kidney Liver Muscle Aflatoxicol
(?g/kg)

Aflatoxin B1
(?g/kg)

Aflatoxin M1
(?g/kg)

4.47 1.68 0.26 0.17 0.06 0.02 0.05 0.01

23.6 36.5 4.7 3.0 2.9 1.2 0.6 0.3

11.2 6.0 5.8 3.9 1.3 0.8 0.4 0.1
Truckses et al. 1982

35 Table 10 – Quantified aflatoxin concentrations in organs of swine intoxicated for 35 days with different levels of aflatoxins in their diets.

Experimental Groups Tissue Liver Toxin M1 ?g/kg B1 ?g/kg B2 ?g/kg Kidney M1 ?g/kg B1 ?g/kg B2 ?g/kg Muscle M1 ?g/kg B1 ?g/kg B2 ?g/kg Adipose Tissue M1 ?g/kg B1 ?g/kg B2 ?g/kg 9 ?g/kg of feed (90% B1 and 10% B2) 0.071 0.002 0.000 0.166 0.027 0.000 0.000 0.010 0.001 0.000 0.000 0.000 524 ?g/kg of feed (90% B1 and 10% B2) 1.479 0.484 0.053 3.132 0.681 0.138 0.206 0.210 0.027 0.010 0.030 0.000
Beaver et al.1990.

Pathogenesis Aflatoxins B1, B2, G1 and G2 have slightly different chemical structures which interact with different organic molecules of the intoxicated animal. Aflatoxin B1 is the most prevalent and the most toxic of the group, therefore, more emphasis has been put on its study, and the majority of the noted biochemical effects refer specifically to this toxin. The aflatoxin molecule is activated metabolically before producing its acute and chronic effects. The activation occurs in the microsomal system of the liver, mediated to a large extent by cytochrome P450. The activated metabolites are capable of interacting with macromolecules and organelles. The enzymatic hepatocyte system facilitates hydroxylation, dimethylation, reduction, and epoxidation that occur during the process of biotransformation. The reciprocal action between these types of activated molecules and the hepatic cells

36 apparently occurs in different sites. When it occurs in the nucleus, it inhibits RNA polymerase, DNA dependent, and the toxin bonds covalently to DNA, and in the exterior of the nuclear membrane, inhibits RNA synthesis by inhibiting RNA enzyme polymerization. Aflatoxin increases the permeability of mitochondria and interrupts the transport of electrons, diminishing or completely inhibiting cellular respiration. Simultaneously, the lysosomal membrane increases its permeability, interfering in the transportation and exchange of nutrients and metabolites. The activation of the lysosomal enzymes and their intracellular extravasation is one of the highly toxic elements of aflatoxins, given their deleterious effects on cellular structures constitute a very harmful component in the pathological process. Yet, significant alterations occur in the endoplasmic reticulum which culminate with the degranulation of the ribosomes, inhibiting diverse metabolic functions such as protein and enzyme synthesis, and the synthesis of coagulation factor II and VII. The main metabolites formed by aflatoxins are aflatoxin M1, aflatoxin Q1, aflatoxin B2a, aflatoxin B1 2,3 epoxide, aflatoxicol, aflatoxicol M1 and aflatoxicol H1. Aflatoxin B1-8,9 epoxide is the most toxic metabolite, and it is responsible for the alkylation of nucleic acids, and subsequently the activation of carcinogenic capacities. The effects of aflatoxins on the different enzymatic systems, membranes, and organs compromise the lipids, carbohydrates, proteins, and nucleic acids metabolism. The interference in the metabolic process of lipids starts at the intestinal absorption level, which decreases by the interference in the excretion of lipases and bile. Furthermore, aflatoxin Aflatoxins decrease the absorption of nutrients.

induces the formation of peroxides, starting from lipids, such as hydroxyl free radicals (OH-), superoxide O2- and nitrogen peroxide in the liver, which have significant toxic effects on proteins, enzymes, and nucleic acids. Two types of interactions between aflatoxins and nucleic acids are known. One of these is the result of a weak, non-covalent and therefore reversible bond. The other, is a covalent, irreversible bond forming aflatoxin-DNA adducts. The main carcinogenic effects are the result of the biotransformation of aflatoxin B1, which is biologically inactive in its natural state. Aflatoxin B1-8,9-epoxide in mammals is the active carcinogenic form. It possesses highly nucleophilic properties, with a high affinity to

37 establish bonds with nucleophilic sites of macromolecular components, establishing very strong (covalent) chemical bonds with diverse molecules such as proteins, RNA, and DNA. In this way, adducts which represent the initial toxic effects of aflatoxins, which can inclusively induce neoplasias, are formed. Adducts are formed by the bonding of activated molecules (epoxides) with the basis of the DNA molecule, specifically in the N7 position of codon 249 of the tumor suppressor gene. The points of DNA mutation can be formed by removing the bonding points formed by the adducts, or by the transvertion of the basis, which induce and promote the formation of cancer. Hydroxylated derivatives of aflatoxin B1 have a significantly lower toxicity because they do not have the properties to establish covalent bonds and adducts with nucleic acids. The toxicity of aflatoxins depends on the formation of aflatoxin epoxides, for which it is necessary that carbons 8 and 9 of the aflatoxin molecule be unsaturated. Since aflatoxins B2 and G2 do not posses these unsaturated carbons, they are considered practically non-toxic compared to aflatoxins B1 and G1. A large part of the toxic effects of aflatoxins occur in the hepatocytes, probably because they are the cells with the highest metabolic activity, as well as possessing high concentrations of cytochrome P450 enzymes, which are responsible for the production of the active compounds derived from aflatoxins. Knowledge of the carcinogenic effects of aflatoxins in experimental animals, and chronic exposure in humans has lead to the general consensus that aflatoxin is associated to the development of hepatocellular carcinoma, one of the most frequent forms of cancer in the world. Even though the liver is known as the main organ affected by aflatoxins, respiratory exposure to aflatoxins by means of dust inhalation, significantly increases the incidence of cancer in the respiratory tract, especially in the lungs of animals and humans. Clinical signs The clinical signs and the severity of aflatoxicosis can vary according to the age of the animals (fetuses and younger animals are most sensitive- Figure 6), type of aflatoxin (B1 is the most toxic), diet composition (diets with high protein levels partially protect animals from the toxic effects), duration of exposure to the toxin, nutritional status, and, above all, on the aflatoxin concentration in feed. Thus, aflatoxicosis can occur in two clinical forms: acute

38 and chronic. Both are dependent on the dose and duration of exposure. Acute toxicosis is quickly recognized by hepatic lesions, clinically characterized by depression, anorexia, icterus, and hemorrhages. In super acute cases, the clinical signs are seen in approximately 6 hours and quickly result in death. In less serious cases, clinical signs begin to appear between 6 and 12 hours after ingestion, a rise in corporal temperature (40.0 to The recuperation rate for swine intoxicated by aflatoxins is low.

41.1°C) can be detected, accompanied by muscular tremors and problems in motor coordination caused by hemorrhages that occur in ham musculature. Diarrhea is often present, demonstrating poorly digested feed, dark green in color and generally liquid (Figure 7).

Figure 6 – Abortion induced by aflatoxin intoxication. (Migliavaca & Prof. Driemeier)

Figure 7 – Paste-like feces with poorly digested feed particles resulting from aflatoxin intoxication

It is common to observe rapid involution of the mammary gland and an overall poor condition of lactating sows. After 24 hours, swine can present hemorrhagic and diphtheric diarrhea. Swine that surpass the clinical symptoms of acute aflatoxicosis, generally no longer recuperate lost weight or overall health. Rather, they remain thin with poor feed conversion, even after a long period following intoxication.

39 The importance of aflatoxins in swine production is not only due to the damages caused by the clinical symptoms of acute intoxication. The main economic losses are due to the ingestion of low concentrations of aflatoxins, which causes chronic intoxications and represent more than 90% of all cases. Hence, the main concern for producers should be subclinical intoxications because the level of aflatoxins found in feed is More than 90% of aflatoxin intoxications are chronic.

generally not enough to produce the perceivable clinical symptoms, but it is enough to significantly decrease productivity. Chronic aflatoxicosis occurs when toxins are ingested during prolonged periods in small doses and concentrations smaller than 1 mg/kg of feed. In these cases, it is common to see loss of appetite, lethargy, decrease in weight gain and general sick appearance. It is sometimes followed by diarrhea several weeks after the initial consumption of contaminated feed (Table 11). Yet, clinical signs are milder and present slow evolution. Scaly skin, curly and opaque hair, areas of red and purple coloring of the skin, lethargy, and depression can be observed. A decrease in feed consumption, decrease in weight gain, worsening of feed conversion, as well as a decrease in animal productivity can also be observed. At the final stages of toxicosis, there are frequent signs of ataxia, icterus, and sometimes convulsions. Because symptoms are often not perceived until later signs appear, and because of the difficulty of clinical diagnosis, the economic consequences can be particularly severe. Furthermore, chronic intoxication is often inadequately diagnosed; often confused with problems in management, nutritional deficiencies, genetic quality of the animals, or confused with opportunistic infections which plague the animals as a result of the chronic aflatoxicosis itself. Because of the heightened difference in sensibility to aflatoxins that exists in different animals, even when the lot is made up of animals of the same descendancy, lack of uniformity in development is inevitable, forming the so called “uneven herd” in terms of weight and size of the animals (Figure 8 and 9).

40 Figure 8 – Comparison of the development of two pigs. 0 ppm of aflatoxin (control) and 1 ppm of aflatoxin in feed for 21 days.

Figure 9 – Lack of uniformity in the development of pigs after an aflatoxicosis outbreak.

.

41 Besides provoking hepatotoxicosis with the proliferation of bile ducts, hepatic steatosis, and hemorrhages, aflatoxicosis produces immunosuppression, is highly mutagenic, teratogenic, and carcinogenic. In the most severe cases, when the illness reaches the terminal stages, there are pathological alterations characterized by icterus in the mucosa membranes and in the subcutaneous tissues, as well as diffused hemorrhages, especially in the kidneys and gastrointestinal tract. Intra-abdominal hemorrhaging and/or hemorrhaging in the ribcage can also occur. Table 11 – Clinical symptoms produced by different levels of aflatoxins in adult pig feed.

Levels (?g/kg) 20-150 150-300 200 400-500 810 450-1,500 2,000

Clinical symptoms Decrease in productivity and lack of uniformity. Decrease in weight gain and increase in susceptibility to diseases. Poorer feed efficiency. Organ lesions and variations in levels of serum proteins. Increased mortality. Reproductive failures. Mortality occurs within 3 days.

Although the literature suggests that chronic intoxications in swine occur with concentrations of toxins less than 1 mg/kg, there is a consensus amongst scientists, which is confirmed by recent findings, that ingesting levels as low as 10 ?g of total aflatoxins per kg of feed can cause significant decrease in swine productivity. These effects can be clearly seen in decreased feed consumption and consequently a decrease in weight gain, as well as a decrease in the average number of pigs born per sow per year. Clinical Pathology Hepatic lesions, besides producing a decrease in protein synthesis, increase the activity of aspartate aminotransferase, gamma-glutamyl transferase, and alkaline phosphatase enzymes. The increase in activity of hepatic enzymes such as arginase, sorbitol

42 dehydrogenase, and alanine aminotransferase is an indication of hepatic lesion in different species, therefore being valuable in diagnosing aflatoxicosis. Sorbitol dehydrogenase, an enzyme of the cellular cytoplasm, is indicative of hepatic lesion. However it is not commonly used in diagnostic exams because it is only present for a short period in the blood stream. Alanine aminotransferase is the preferred diagnostic tool, but is an enzyme that is found in low quantities in swine, thus being difficult to detect. Therefore, some researchers did not find significant alterations of this enzyme in swine intoxicated by aflatoxins. The serum values of aspartate aminotransferase are more sensitive to intoxication by aflatoxins, showing a significant increase of activity approximately 14 days after the initial intoxication. Alkaline phosphatase is an enzyme that is present in various types of tissues, but it is only important in diagnosing alterations of the hepatic and bone tissues. With the exception of growing animals or those that have bone lesions, the elevation of this enzyme has a hepatobiliary origin. Cholestasis (blocked biliar flux) brings on elevated serum levels of alkaline phosphatase. Various researchers found significant increases in enzymatic activity after pigs were intoxicated with aflatoxins at rates of at least 450 ?g/kg in feed. Gamma-glutamyl transferase is an enzyme associated with the membrane of various tissues, therefore, it is an important indicator in the process leading to cholestasis. It can be highly concentrated on the edges of epithelial cells of the kidney and bile ducts because increased serum concentrations always originate in hepatobiliary tissues. Elevations of the enzyme were not observed in pigs intoxicated with 1.2 mg of aflatoxin/kg of feed for a period of 72 hours. This can be explained by the fact that gamma-glutamyl transferase only increases significantly following ciliary necrosis, severe cholestasis, and/or hyperplasic nodules. Researchers found an increase in gamma-glutamyl transferase in pigs intoxicated with 4 mg of aflatoxins/kg of feed given ad libitum for a length of 14 days. Concentration of 3 mg of aflatoxins/kg of feed only increased serum activity to a significant level after 28 days. Pyruvic glutamic transaminase enzyme always presents significant increases, doubling or tripling its activity after animals are intoxicated by aflatoxins. Oxaloacetic glutamic transaminase enzymes and ornithine carbonyl transaminase are not good indicators in diagnosing aflatoxicosis outbreaks because they present a rapid increase in activity, generally between 12 and 24 hours, and their decrease is equally fast.

43 It is normally recommended that a combination of enzymes be used to diagnose aflatoxicosis in swine. Therefore, alkaline phosphatase (high sensitivity), aspartate aminotrasferase (moderate sensitivity), and alanine aminotrasferase (low sensitivity) are recommended. The prothrombin time, approximately 20 seconds in swine, increases to approximately 60 seconds between 12 and 24 hours after intoxication. A decrease in plasmatic proteins always occurs after a period of intoxication due to hepatic damage. Macroscopic Lesions Death generally occurs in a period of 3 days when there is acute aflatoxicosis. Immediate changes evident in clinical exams are pale mucosa, and a bulging abdomen caused by the decrease of feed intake, dehydration, and gastric deflation caused by diarrhea. The presence of liquid feces can normally be found in the perineal area because poorly digested feed are eliminated in abundance, even while the animal is lying down dying. The liver is always the most affected of all the organs because alterations start occurring from the moment of ingesting contaminated feed. After 6 hours of acute intoxication, the liver presents a pale-bronze color and appears as though it had been cooked. Twelve hours after intoxication, the liver presents hemorrhaging sites on the surface with diameters of approximately 1mm. Soon after, the liver becomes pale, edema become present, and the liver becomes friable. The gall bladder displays thickening of the walls, induced by edema with the formation of fluid in the submucosa, muscle, and serosa. The heart can exhibit areas of hemorrhaging, principally affecting the subepicardial and endocardial regions. Extensive enterocolitis can occur in the intestine, but it is most frequently present in the jejunum and the ileum, where it is possible to see disseminated hemorrhaging with free blood in the lumen. Generally, the cecum and the colon become hyperemic, with different quantities of blood on the outside. A change in liver color is one of the first signs that an animal is intoxicated with aflatoxins. The prothrombin time could increase when there is intoxication by aflatoxins.

44 Acute intoxications can lead to the formation of fluid accumulation in different cavities of various organs. These pools of fluids are most frequently found in the heart and the peritoneum. Jaundice in various organs and tissues can be present, especially when the intoxication becomes subacute or chronic. Jaundice of the carcass, associated with a swollen and yellow liver, are very strong indicators of intoxication (Figure 10). Figure 10 – Hepatic changes in growing swine after 21 days of intoxication with aflatoxins. (Colum A) Normal livers (Colum B) Livers after intoxication with 1.0 mg of aflatoxins per kilogram of feed.

A

B

Chronic exposure to low doses of aflatoxin results in jaundice in the whole carcass. The liver can display a pale-yellowish appearance with very well defined lobes, hemorrhaging sites, especially in the parietal surface (Figure 11), and has different levels of fibrosis and cirrhosis. In the case of cirrhosis, a pale liquid (straw yellow) can be found in the abdominal and thoracic cavities, and accumulation of the liquid in the lungs leads to pulmonary edema. Aflatoxicosis = jaundice + distorted liver.

45 In these conditions, the gall bladder generally appears edematous, an increase in size, with thicker content than usually found in healthy animals. Jaundice tends to be generalized, accompanied by yellowish coloration of body fluids, which can be located in various areas, even reaching the thoracic and abdominal cavities where Aflatoxins levels lower than 10 ppb in feed are considered safe.

the fluids concentrate in the spiral colon. There is a tendency of yellow liquid to accumulate in the pericardial sac of the thoracic cavity. There is also a decrease in coagulation time, therefore, it is possible to see an accumulation of blood in cavities and mucosa, other than hemorrhages in muscular tissue. Figure 11 – Hepatic lesions caused by aflatoxicosis in pigs. (A) Pale-yellowish quality with very well defined lobes and hemorrhaging sites. (B) Red and yellow areas, giving a nutmeg appearance (Migliavaca and Prof. Driemeier).

A

B The kidneys generally become swollen or hypertrophied and pale in color, depending

on the degree of intoxication. Some researchers indicate the possibility of finding formation of areas of consolidation in the pulmonary parenchyma. DL50 of aflatoxins is very low (0.62 mg/kg) in pigs, and 50 ?g/kg (ppb) of feed is considered a safe dose. However, because of the difficulty of sampling feed correctly, this level of toxin is regarded as being very high. Lower levels continue to be important in inducing immunosuppression, which can sometimes lead to clinical symptoms which are difficult to correlate to aflatoxin intoxication.

46 Microscopic Lesions The principal toxic effects of aflatoxicosis are observed in the liver. Acute intoxications produce evident alterations, such as disruption of cell cords and granular cytoplasm with vacuoles, few hours after intoxication. Then, there is hypertrophy of cells leading to stenosis of the sinusoids and the appearance of necrotic foci randomly distributed in the parenchyma. During this evolutionary stage of lesions, compromised nuclei are not very evident. Notwithstanding, after 12 hours, necrotic foci with diameters greater than 100 ?m can abound when there is a moderate to severe intoxication. After 24 hours, changes are even more evident: there is evident hepatocyte necrosis, hemorrhaging in the perisinusoidal space (Disse’s space), and, above all, centrilobular congestion. Other changes, such as cellular infiltration in the centrilobular region, as well as edema, and biliary stasis associated to hyperplasia and the proliferation of bile ducts, mainly observed in subacute and chronic toxicosis, can also be present. With the chronicity of aflatoxicosis, fatty infiltration, accumulation of glycogen in hepatocytes and fibrosis, and centrilobular necrosis occurs. Intestines are almost always affected. Enteritis can be acute and there can be hemorrhaging with necrosis of the villi. In the most severe cases, necrotic debris from the intestinal mucosa can be found in the intestinal lumen. The lamina propia and submucosa normally present high concentrations of lymphocytes, granulocytes, pasmocytes, and macrophages. The presence of carcinomatous lesions are frequently observed in adult animals that consume feed contaminated with low concentrations of aflatoxins, starting at 10 ?g/kg of feed for periods of approximately 24 months. Epidermoid and liver carcinomas exhibit metastases in surrounding tissues such as omentum and hepatic lymph nodes. However, the most frequently observed microscopic changes in the liver are diffused centrilobular necrosis, fat degeneration, and different levels of hemorrhages. In subacute cases, cellular necrosis of the hepatic parenchyma is not as prominent, but there is marked proliferation of bile ducts, fibroses, and conjunctive tissue in the interior and between hepatic lobes, which creates something like a post-necrotic cirrhosis.

47 Aflatoxin effects on the immune system The immunosuppressant effects of aflatoxins have been demonstrated in laboratory and domestic animals, especially poultry. Although there is a consensus concerning immunotoxicity, its mechanism is still not completely clear. The effects that mycotoxicosis has on the complement, interferon and concentration of serum proteins are the result of hepatic damages and decrease of protein production. Other than compromising the formation of interferon and complement, it is known that aflatoxins decrease phagocytic capacity of macrophages and the migration of leukocytes and lymphocytes. They also cause aplasia of the thymus and a decrease in weight of the bursa of Fabricius in poultry, thus, principally affecting immunity. Aflatoxin B1 principally affects T lymphocytes, including T auxiliary cells, and T suppressant cells. The effects of aflatoxins on immunoglobulin are also not entirely clear. Various studies indicate a decrease in IgA and IgG levels of animals intoxicated with feed containing more than 500 ?g/kg of aflatoxins. However, other studies concluded that an increase in immunoglobulin concentration such as IgG can occur, and they attribute this to the inability of the damaged liver to remove the antibodies produced in the gastrointestinal tract. The clinical effects of aflatoxins in swine can also be detected by the decrease of immunity and increase of secondary diseases in swine severely affected by aflatoxicosis. Various researchers found the co-occurrence of clinical outbreaks of aflatoxicosis with an immediate increase in septicemia infections caused by different bacteria, such as: Salmonella, Pasteurella and Erysipelothrix. Failure or decrease in the effectiveness of vaccines in different manifestations of aflatoxicosis in the animals. The severity of aflatoxin effects on the immune system does not only depend on the levels of aflatoxins present in the feed consumed by pigs, but also on the nutritional status and general health of the animals. The presence of other illnesses or the ingestion of other toxins can exert an interactive influence on the immunological response of the intoxicated animals. Several researchers have demonstrated that a healthy diet decreases the quantity of toxic Failures of vaccines can be attributed to aflatoxicosis. Aflatoxins affect the swine immune system.

species has been frequently related to the presence of aflatoxins in feed, including clinical

48 residue in the organs and it significantly improves the function of different body organs. An increase of protein and vitamins in diets, particularly vitamins B12 and K, improve the animals’ resistance against the effects of intoxication. Vitamin K, particularly, decreased protombine time and prevented hemorrhages. Lipotropic agents such as methionine have the important function of improving metabolism of fatty acids in the liver, reducing the severity of fatty degeneration. Diagnosing Aflatoxicosis The clinical symptoms of acute aflatoxicosis are easy to diagnose. The introduction of a new batch of feed, often with altered macroscopic characteristics, has been historically associated with the appearance of symptoms. Clinical symptoms as well as anatomical and pathological findings after changing feed, suggest intoxication by aflatoxins. The greatest difficulty in diagnosing aflatoxicosis is presented by the period of time in which clinical symptoms appear which can take two or more weeks, considering the concentrations usually present in commercial feed. In hematological evaluations, there appears to be an increase in the activities of alkaline phosphatase, aspartate aminotransferase and oxaloacetic transaminase. Chronic intoxications tend to go unnoticed in clinical evaluations of animals. In these cases, there is a loss of productivity that becomes significant when assessing the productivity of the farm. The safest assessment used to diagnose aflatoxicosis consists in identifying aflatoxin present in feed using chromatographic methods such as Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC), and mass spectrometer such as GC/MS, HPLC/MS e HPLC/MS/MS, which are more efficient. Immunological methods, such as ELISA kits, have shortcomings; therefore, they are only used as semi-quantitative methods. The sample being evaluated should be representative of the batch, and must follow the technical procedures regarding the method of sampling collection.

49 Prophylaxis and Treatment The treatment of mycotoxicosis represents one of the greatest challenges for clinical veterinary. It is therefore very important that this problem is solved by intervening in multiple processes. Considering the fact that mycotoxins are natural occurring substances in practically all food sources; they do not present immune response, and their toxic and economic effects emerge only after a determined period of ingestion (which could be a few days or hours in acute cases or weeks in chronic cases) many alternative treatments should be considered. Measures related to inhibiting and decreasing fungal development and subsequent formation of mycotoxins during farming, as well as in storage, deserve special attention. Preventing the formation of mycotoxins should always be one of the principal measures adopted. Storing feed where there is excess humidity, in silos that are poorly sealed and therefore prone to leakage and infiltration, should be avoided. The use of organic acids can help to preserve feeds in conditions favorable for mold development. Constant and continual monitoring of mycotoxins in feed production, including appropriate sampling techniques, is the most efficient technical option, and from an economic standpoint, most viable, especially for medium to large sized swine producers. Knowing the concentrations and frequency of aflatoxins present in diets, allows producers to make a better decisions to efficiently use absorbents to prevent and control aflatoxicosis. Due to the high sensitivity of swine to the majority of mycotoxins, especially aflatoxins, absorbents should be used when more than 50% of samples are contaminated and there is a concentration higher than 10 ?g/kg. This recommendation should be considered in relation to factors such as environmental conditions, age, or diet, given each particular case. Some of the absorbents evaluated in LAMIC/UFSM display the capacity of absorbing aflatoxins in vitro more than 90%, which should be the minimum criterion for the employment of a product in order to avoid or ameliorate the toxic effects of aflatoxins in pigs.

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54 SOARES, L.M.V.; RODRIGUES-AMAYA, D.B. Survey of aflatoxins, ochratoxins A, zearalenona and sterigmatocystin in some Brazilian foods by using multitoxin thin layer chromatographic method. J Assoc Off Anal Chem Int, v. 72, p. 22-6, 1989. SOLOVEY, M.M.; SOMOZA, C.; CANO, G.; PACIN, A, RESNIK, S. A survey of fumonisins, deoxynivalenol, zearalenone and aflatoxins contamination in corn-based food products in Argentina. Food Addit Contam, v.16, n.8, p. 325-9, 1999. SWENSON, D.H.; LIN, J.K.; MILLER, E.C. Aflatoxin B1–2,3-epoxide as a probable intermediate in the covalent binding of aflatoxins B1 and B2 to rat liver DNA and ribosomal RNA in vivo. Cancer Res., v. 37, p. 172-81, 1977. TRUCKSES, M.W.; STOLOFF, L.; BRUMLEY, W.C.; WILSON, D.M.; HALE, O.M.; SANGSTER, L.T.; MILLER, D.M. Aflatoxicol and aflatoxins B1 and M1 in the tissues of pigs receiving aflatoxin. J. Assoc. Off. Anal. Chem., v. 65, n.4, p. 884-7, 1982. TUNG, H.T.; DONALDSON, W.E.; HAMILTON, P.B. Effects of aflatoxin on some marker enzymes of lysosomes. Biochem. Biophys. Acta., v. 222, p. 665-7, 1970. VARGAS, E.A.; PREIS, R.A.; CASTRO, L.; SILVA, C.M. Co-occurrence of aflatoxins B1, B2, G1, G2, zearalenone and fumonisin B1 in Brazilian corn. Food Addit. Contam., v 18, n. 11, p. 981-6, 2001. WILSON, D.M.; SANGSTER, L.T.; BEDELL, M.B. Recognizing the signs of porcine aflatoxicosis. Vet. Med. v. 7, p. 974-6, 1984. WOGAN, G.N.; EDWARDS, G.S.; NEWBERNE, P.M. Structure-activity relationships in toxicity and carcinogenicity of aflatoxins and analogs. Cancer Res. v. 31, p. 1936-42, 1971. WONG, Z.A.; HSIEH, D.P.H. Aflatoxicol: major aflatoxin B1 metabolite in rat plasma. Science, v. 200, n. 4339, p. 325-7, 1978. WORLD HEALTH ORGANIZATION. Mycotoxins Environmental Health Criteria, 11, Geneva, WHO, 1979, p. 21-84. WYAT, R.D. Poultry. In: SMITH, E.J.; HENDERSON, E.R. (eds). Mycotoxins and animal foods. Boca Raton CRC Press, 1991. p. 553-612. YOSHIZAWA, T.; YAMASHITA, A.; CHOKETHAWORN, N. Occurrence of fumonisins and aflatoxins in corn from Thailand. Food Addit. Contam., v. 13, n. 2, p. 163-8, 1996.

55

_________________ 3 ____
ZEARALENONE

56 ZEARALENONE Summary Zearalenone is a non steroid estrogenic fungal metabolite, chemically describe as a phenolic resocyclic acid lactone. It has good thermal stability and low solubility in water, but it is highly soluble in organic solvents. It is produce by various fungal species of the Fusarium genus, including F. culmorum, F. graminearum and F. crookwellense. These species colonize cereals and tend to become particularly important during periods of high humidity, accompanied by mild temperatures. Thermal oscillation within low temperatures is favorable for the production of large quantities of this mycotoxin. Temperatures around 25oC are favorable for fungal growth. A decrease in temperature to approximately 10oC with humidity higher than 14% triggers a secondary metabolism responsible for zearalenone production. For this reason zearalenone is a natural contaminate of cereals such as wheat, barley, rice and particularly corn, in different countries around the world. When contaminated cereals or their byproducts Zearalenone intoxication is generally manifested when concentrations surpass 100 ppb.

are ingested by different animal species, the toxin can produce estrogenic effects. Among domestic animals, swine are the most sensitive, showing clinical symptoms of intoxication starting at 0.1 mg of toxin/kg of feed consumed. Bovines can present problems related to infertility starting at 14 mg of zearalenone/kg of ingested feed. When swine consume diets contaminated with zearalenone, clinical symptoms characterized by vulvovaginitis, birth of weak piglets and stillborns, and outbreaks of splayleg often occur. There is also an obvious decrease in conception rate accompanied by repeated heat. History The first reported case of hyperestrogenism syndrome in swine occurred in the USA in 1928. However, a correlation between cause and effect of the outbreak was never established. Starting in 1952, the syndrome was associated with the incidence of fungi of the Fusarium genus in the feed consumed by the affected animals. After more than 10 years of research, it was established that a toxin produced by the fungi of the Fusarium genus caused the disease, and it was then named zearalenone or F-2

57 toxin. After this initial report, the syndrome was identified in various European and Asian countries, Australia, Canada, and Brazil in 1985, where the syndrome was diagnosed in various swine operations in the state of Rio Grande do Sul. Etiology Zearalenone, also known as F-2 toxin, gets its name from the producing fungus in its perfect state (sexual) Giberela zeae (Fusarium graminearum = Fusarium roseum). However, many other fungi such as F.avenaceum, F.culmorum, F.cerealis, F.equiseti, F.moniliforme, F. semitectum, F.sporotrichiodes and F.oxysporum can produce the toxin. Zearalenone is a phenolic resocyclic acid lactone (Figure 12), classified, according to its biosynthetic origin, as a nonoketide within the poliketides group. The majority of zearalenone can be produced by Fusarium, but only trans-α-zearalenol [6-(10-hydroxy-6-R-hydroxy-trans-1-undecenyl)-βresocyclic-acid-lactone] occurs naturally in cereal grains, as a white crystal compound with a relative molecular weight of 318, a melting point of 164-165°C, and maximum absorption (and coefficient of absorption) in 236 nm (29,700), in 274 nm (13,909), and in 316 nm (6,020). Zearalenone is blue-greenish fluorescent when excited with long UV wavelength (360 nm), and a more intense green when excited with short UV wavelength (260 nm). Like most mycotoxins, zearalenone also displays good thermal stability, thus undergoing little damage during the process, preparation and/or harvesting of feed ingredients. The level of toxin does not decrease when purified aliquot is submitted to 120°C for a period of 4 hours. The toxin displays stability when present in crushed corn at 150°C for a period of 44 hours, proving that zearalenone is only partially decomposed by heat during the processing of contaminated cereal. Crushing and extrusion processes carried out at 140?C also entail significant degradation of zearalenone levels in cereals. The processing of contaminated rice can significantly decrease the presence of zearalenone, up to a 37% reduction in cooked rice. Approximately 60% of zearalenone remains during the manufacturing of breads made with contaminated ingredients, around 40-50% persists in the preparation of pasta, and 80% endure during the fabrication of cookies. The presence of zearalenone has been detected in beer produced in different countries, despite the fact that the fermentation process reduces levels of contamination by approximately 50%.

58 Figure 12 – Chemical structure of zearalenone and some derivatives.

OH

O

CH3

Y OH K X

X =O =O =O OH OH

Y H OH H OH H

K H H CHO H H

Name Zearalenone 8`-hydroxyzearalenone 5-formilzearalenone 6`,8-dihydroxyzearalenone Zearalenol

Occurrence of zearalenone in feed and food The presence of zearalenone in feed and feed ingredients is mostly limited to regions that have favorable climatic conditions, especially low temperatures (8 to 14oC). There is a consensus that temperature oscillation, from low to moderate (8 to 24oC), is essential for the formation of significant quantities of zearalenone. Other factors that have an important role in the production of zearalenone are high humidity of the substrate, for it is well known that fungi of the Fusarium genus require high humidity levels to develop. The highest quantity of toxins is produced when the percentage of water in cereal is greater than 22%, or when water activity is at approximately 0.85% or higher. The pH of the substrate is of little importance in the quantity of toxin produced, fluctuating between 3.5 and 7.5, without exhibiting significant differences in productive crops. It is common that cereals are transported over great distances, and that grains are imported from other regions and countries, therefore, clinical symptoms related to zearalenone have been observed in tropical climates.

59 Zearalenone occurs in a large part of agricultural products, grains, and feed, including corn and its byproducts, breakfast cereals, beer, wheat flour, breads, nuts, and a variety of other products consumed by animals and humans. Although natural contamination occurs in different grains, it is most prevalent in rice, oats, barley, corn, sorghum, wheat, and animal feed produced with these grains. Evaluation of zearalenone contamination indicated 20 to 37% positivity in Germany, 41.7% in England, 1% in Holland, 30% in Egypt, and 12.5% in South Africa. In Colombia 55.6% of sorghum, 6% of corn, 31% of rice bran, and 37.5% of swine feed samples were positive for zearalenone contamination. In some countries, zearalenone was found in corn flour and breakfast cereals destined for human consumption, at levels of up to 70 ?g/kg, correspondent to concentration levels about 400 times lower than those that cause acute effects in monkeys or rats under experimental conditions. In certain parts of Africa, substantially higher levels were occasionally found in beer and in macerated liquids prepared with contaminated corn and sorghum. In Zambia, the toxin was found in different raw materials and foods, such as cornflakes, beer, and corn at concentration levels which varied from 100 to 4600 ?g/kg. Concentrations in foods and feed vary greatly depending on climatic conditions. Zearalenone was found in 11 to 80% of wheat samples, and in 7 to 68% of barley samples collected in Southeast Germany in 1987, and from 1989 to 1993, respectively, with average annual indices of 3 to 180 ?g/kg in wheat (highest concentrations of 8,000 ?g/kg) and from 3 to 36 ?g/kg in barley (highest concentrations of 310 ?g/kg). After the 1995 harvest, year marked by heavy rains, 140 samples of wheat for human consumption were taken from all regions of Bulgaria and there was a 69% frequency of zearalenone contamination, with an average contamination of 17 ?g/kg to up to 120 ?g/kg in positive samples. The occurrence of zearalenone has been amply studied in France, presenting high indices of the toxin, but in low concentrations. In studies done in the USA, zearalenone was found in 6 of the 576 samples analyzed, with levels which varied from 450 to 800 ?g/kg. In 1972, when climatic conditions were very favorable for Fusarium development, zearalenone was found in 17% of the 223 samples of corn, with concentrations that oscillated between 100 and 5,000 ?g/kg. In studies done in Buenos Aires and in the province of Santa Fe (Argentina) from 1983 to 1994, zearalenone was found in 30% of the 2,271 samples of corn collected, with an average concentration of 165 ?g/kg (annual variation between 46 to 300 ?g/kg) and a

60 maximum of 2,000 ?g/kg. Concentrations in ecologically grown wheat and rye were higher than those grown with normal intensive cropping. The toxin was found in 40 of the 201 grain samples, with average concentrations of 24 ?g/kg in wheat and 51 ?g/kg in rye in crops that were planted and cultivated with alternative methods, and 6 ?g/kg in wheat and 4 ?g/kg in rye in conventional crops. The highest concentration of zearalenone was of 199 ?g/kg, which was found in rye cultivated with alternative methods. The occurrence of zearalenone in byproducts of grains and fermented grains has also been widely studied in different countries. The toxin is present in approximately 10% of fermented beverage samples, including beer, with concentrations between 8 and 53 ?g/L. Zearalenone is also frequently detected in silage made from grains and pasture, occurring principally in silage made from raw-materials after the process of maturation. Concentrations generally reach levels of up to 300 ?g of toxin/kg of silage. The concurrent occurrence of zearalenone and its derivatives and other mycotoxins is very frequent because they are produced by the same molds that produce deoxynivalenol and nivalenol. In Argentina, various studies showed contamination of zearalenone, varying from 1 to 30% of the total samples. In Brazil, 20% of 105,509 samples analyzed by LAMIC/UFSM, collected to monitor the feed industry, were contaminated. Although the average positivity is low, in certain periods of the year, the rate of positivity surpasses 45% of analyzed samples. In this laboratory, the was 18.6 mg/kg. Toxicokinetics of zearalenone The rate of absorption of zearalenone in swine gastrointestinal tract has not been well established. It is believed, however, that around 80 to 85% of the toxin and/or products of its biotransformation are readily absorbed. Zearalenone can be biotransformed immediately after ingestion by intestinal microflora or by mucosa cells. Quickly following this, compounds are bond to endogenous substances and distributed through the bloodstream. It is also thought that there is enterohepatic circulation of the toxin because the toxin can be detected in feces for the Around 20% of feed samples analyzed in Brazil are contaminated with zearalenone.

average concentration of zearalenone found was 74.1 ?g/kg, and the maximum level detected

61 exact amount of time as it can be detected in the blood. The biotransformation of zearalenone occurs mainly in the liver. In mammals, it creates different steroisometric compounds, especially α and β-zearalenol, by the reduction of the ketone grouping in C6. These compounds immediately bind to glucuronic acid. Another similarly structured compound is zeranol, which is synthetically produced from zearalenone and used to stimulate weight gain in animals. It differs from zearalenone because of the absence of a double bond between C1 and C2. The proportions between the products of biotransformation and zearalenone are different amongst different intoxicated animal species. It is thought that this is fundamental in explaining the difference in susceptibility of species to zearalenone intoxication. Elimination When a moderate dose of zearalenone is administered orally to swine, approximately 45% of the toxin and/or products of its biotransformation can be detected in urine after 48 hours. Approximately 22% of the toxin is retrieved from the feces, combining for a total recuperation of approximately 70% in 48 hours. However, a consistent pattern does not exist because other researchers have concluded that zearalenone or its biotransformation products can be detected up to 5 days after ingestion, estimating a half-life of 87 hours, when a moderate dose of zearalenone is administered orally. Zearalenone can be secreted through the milk of milking cows after being fed with contaminated feed. The maximum quantified concentrations in cow milk, with an oral dose of 6,000 mg (equivalent to 12 mg/kg of live weight), were 6.1 ?g of zearalenone/L, 4 ?g of αzearalenol/L, and 6.6 ?g of β-zearalenol/L. Neither zearalenone, nor its biotransformation products were detected in milk of cows fed with 50 or 165 mg/kg of zearalenone (equivalent to 0.1 to 0.33 mg/kg of live weight) for 21 days. Zearalenone residue in animal tissue The amount of detectable zearalenone in tissue of intoxicated animals varies greatly, depending on the concentration of toxin in the ingested feed, time of exposure, and animal species. Few studies have been developed to determine the quantity of zearalenone and its biotransformation products in organs and tissues of intoxicated animals. However, some

62 results are available: in swine liver, 78 and 128 ?g/kg were detected when the animals consumed feed contaminated with 40 mg of toxin/kg of feed for a period of four weeks. Chicken feed with 100 mg of zearalenone/kg of feed exhibited concentrations of up to 103 and 681 ?g/kg in muscular tissue and liver, respectively. According to the recommendations by the International Committee of Risk Evaluation to Mycotoxin Exposure, the maximum levels of zearalenone residue in foods destined for human consumption are 10 ?g/kg in the liver and 2 ?g/kg in muscular tissue. Because zearalenone can be transferred to beer by contaminated grains in various stages of the fermentation process, incidence of up to 58% and high concentrations of zearalenone were found in these products, including up to 2 mg/L in Nigeria, up to 53 mg/L in Switzerland, and up to 4.6 mg/L in Zambia. In similar studies, zearalenone and α or βzearalenol were not found in Canadian, Korean, or European beers, with the exception of French beer that contained 100 ?g/L. Zearalenone detection in products, such as beer, that undergo a fermentation process should be done by identifying the metabolites, especially α and β-zearalenol. Pathological mechanisms of action of zearalenone The capacity of zearalenone to unleash hyperestrogenism syndrome in swine has been known for many years. Contrary to other species, such as poultry and bovine, α-zearalenol is produced in greater quantities in swine. The reactions of zearalenone biotransformation to zearalenol are catalyzed by 3-α-hydroxysteroid-dehydrogenase enzyme (3-α-HSD) (Figure 13). This enzyme is also known for degrading 5-androstan-3,17-dione, which is a product of steroid metabolism. Swine appear to be the species most sensitive to zearalenone. Thus, uterotrophic activity is attributed to different zearalenone biotransformation products, especially α-zearalenol, which is produced in large quantities in this species. Zearalenone and/or its metabolites interact with estrogenic receptors, and have significant effect on endometrial secretions, uterine protein synthesis, and uterine weight gain. Furthermore, it maintains the corpus luteum in the absence of gestation. Alterations caused by zearalenone in the reproductive tract occur as a consequence of increase in 17-β-estradiol, as well as the decrease in progesterone levels. For this reason, there are alterations in the uterine

63 lumen, caused by changes in endometric secretion activity. Another important effect of zearalenone occurs in the ovaries of young gilts, exhibiting a larger number of first stage atretic follicles. Consequently, there will be less ovulation, fewer number of embryos, and fewer number of piglets born. Toxic effects are not observed in levels below 0.04 mg of toxin/kg of live weight/day, or below 1.0 mg of zearalenone/kg of contaminated feed. However, levels of 250 ?g/kg of feed can reduce the number and weight of piglets born because sows are particularly sensitive to zearalenone during ovulation. There can eventually be abortions or mummified fetuses. International organizations, such as the Joint Expert Committee on Food Additives (JECFA) have established that tolerable levels of zearalenone should obey limits that do not have hormonal effects on swine, which is the most sensitive specie to zearalenone. According to the Committee, the maximum tolerable daily dose for human should be 0.02 ?g/kg of live weight/day. However, more advanced studies are necessary in order to better evaluate the hormonal and possible toxic effects, including zearalenone’s carcinogenicity. Nine countries belonging to the European Union have established zearalenone tolerance limits, mainly in grains, varying from zero to 1 mg of toxin/kg of feed. However, lower concentrations of zearalenone in naturally contaminated foods have already been responsible for causing hyperestrogenic syndrome in gilts, which displayed characteristic signs of the syndrome such as redness, hyperemia and edema of the vulva labia and mammary glands, and vesicular follicles and cysts in the ovaries. Immunosuppressive effects of zearalenone Various studies have been done with the intentions of evaluating the effects of zearalenone on the immune system of different animals. A large part of these studies were done on laboratory animals. Rodents intoxicated by zearalenone did not exhibit a significant increase in susceptibility to infections by Listeria monocytogenes. The animals did have alterations in leukocytes, lymphocytes, neutrophils, monocytes, or eosinophils.

64 Figure 13 – Mechanism of action of zearalenone.

ZEARALENONE 3-αHSD

β-Zearalenol

α-Zearalenol

Concentration

Degradation of H. steroids Cytoplasmic estrogen receptors DNA Proteic synthesis of target cells Hormone secretion Reproductive tract weight HYPERESTROGENISM

Laboratory studies done on cell cultures indicate that zearalenone and its biotransformation products can cause a series of alterations in metabolic processes. Thus, exposure to 14 ?g/mL inhibits DNA synthesis of lymphocytes by 50%. Other studies suggest that zearalenone and its biotransformation products can inhibit mitogenesis that causes a proliferation of lymphocytes B and T. The effects of zearalenone on swine immune system have yet to be fully studied. However, it is thought that its effects are irrelevant compared to the reproductive problems caused by this mycotoxin. Apparently, zearalenone does not affect the immune system.

65 Clinical symptoms Zearalenone and/or its biotransformation products are responsible for

hyperestrogenism syndrome in swine, observed clinically as vulvovaginitis (Figure 14). The clinical symptoms associated with intoxication vary depending on the dose of toxin ingested and the age of the animal (Table 12). Symptoms tend to be more acute the

Clinical symptoms similar to estrus are observed.

younger the animal, as well as the higher the doses of toxin ingested; thus prepubertal gilts, approximately 4 months old, are generally the most severely affected. The initial onset of clinical symptoms varied greatly, appearing between 1 to 4 weeks after the ingestion of contaminated feed. It has been found that young animals are highly sensitive to diverse natural intoxications, in which they exhibit characteristic clinical not affected by that level of toxin. Sick animals display symptoms that mimic estrus, such as reddening and enlargement of the vulva labia, and edema of the mammary glands with a significant increase in volume. At times, animals can display unrest, resulting in fights or cannibalism. Clinical manifestations normally persist for long periods, even after the contaminated feed has been substituted. Often, females’ normal estrous cycle does not return. Vaginal or rectal prolapse (Figure 15), which occurs due to the relaxation of the sphincters, is not always present in contaminated herd. However, it can also be the only evident clinical symptom of zearalenone intoxication. In these cases, secondary infections generally occur, following lesions, and contaminations which occur on the mucosa surface of these organs. Vulvovaginitis is the principal clinical symptom of zearalenone.

symptoms. Adult animals given feed containing the same concentrations of zearalenone are

66 Figure 14 – Zearalenone intoxication. (A) Control gilts (not intoxicated). (B) Gilts with vulvovaginitis after intoxicated with 2 mg of zearalenone/kg of feed for 24 days.

A

B

Figure 15 – Rectum prolapse in swine after being intoxicated by zearalenone.

67 Table 12 – Toxic effects of zearalenone in swine, according to age or stage, associated with the level of toxin in feed. Zearalenone levels mg/kg
3-5

Stage
Prepubertal gilts until first gestation Sows

Clinical symptoms observed
Increase in mammary gland size, edema of the vulva, increase of uterine and ovarian size. (1)

3-5

Increase of uterine size, uterine edema, corpus luteum retention, anestrous for more than 50 days. (1)

Sows Sows 15 days before farrowing Swine 15 days before farrowing Gilts, 20 to 30 kg Gilts, 64 kg Gilts, 64 kg

2.2 5

No clinical manifestations. (4) Weak newborn piglets, edema of the vulva of new born females, and splayleg. (2)

3-4

57% of females are born with hyperestrogenism. (3)

3.5-11.5 3 6-9

Vulvovaginitis and hypertrophia of the reproductive tract. (5) No change in feed consumption. (6) Decrease in daily feed intake and worsening feed conversion. Characteristic signs of hyperestrogenism. (6)

Boar Boar

3-9 2-200

Did not affect libido. (7) Did not affect libido or reproductive potential. (8)

(1) Etienne & Jemmali (1982); (2) Miller et al. (1973); (3) Lancorevic et al. (1977); (4) Shreeve et al. (1978); (5) Farnworth & Trenholn (1983); (6) Young et al. (1986); (7) Young e King (1986) e (8) Ruhr et al. (1983).

Edema of the mammary glands in sows often indicates gestation or labor. Intoxicated animals display an increase of vulva and mammary gland size the last two weeks of gestation, as well as milk in glands at least two weeks prior to labor. Common symptoms in swine intoxicated by zearalenone are: infertility,

pseudogestation, manifestation of permanent estrus, a decrease in the number of piglets born probably due to the decrease of follicle development, embryonic reabsorption, malformations and juvenile hyperestrogenism, which generally occurs in females born from intoxicated sows, a week after being born (Figure 16).

68 Figure 16 – New-born female piglet displaying clinical symptoms of vulvovaginitis.

Hyperestrogenism is characterized by reddening and increase in vulva size, and early development of mammary glands. According to studies done with intoxicated sows, the most critical period of embryonic survival seems to be between the seventh and tenth day of gestation. The probability of abortion in the last two months of gestation is unlikely in swine because estrogens are luteotropic in this species. The refusal or decrease in feed intake is often associated with zearalenone intoxication. This is a result of the bad taste that develops in the feed which is produced by the development and intense fungal contamination, and Decrease in feed intake can be associated with intoxication. Infertility, pseudogestation, and decrease in number of piglets born are associated with zearalenone intoxication.

because of the hepatic damage that this mycotoxin causes. Furthermore, the nutritional value of the grains diminishes, especially energy which is consumed by the fungal population. One of the greatest consequences of zearalenone intoxication in swine is caused by abnormalities in the estrus cycle. Frequently, females exhibit all the typical signs of estrus, but they do not demonstrate sexual receptive behavior, refusing mount. The conception rate of the herd can decrease by up to 70% and pregnant sows generally have small litters. This

69 significantly influence the number of piglets born per sow per year, and the productivity of the farm. The vitality of piglets in the first 24 hours after birth is also severely affected. Consequently, suckling is done with greater difficulty, significantly increasing the number of deaths resulting from crushing and starvation, besides increasing the number of stillborns. The number of viable piglets in the first week of life is also affected. Many piglets display splayleg syndrome (Figure 17), as well as lack of coordination of the limbs. Figure 17 – Piglet born from a zearalenone intoxicated sow, presenting splayleg syndrome.

Intoxicated piglets and females frequently present swollen and reddened vulva labia, and males and females present edema of the mammary glands various days after birth. These clinical symptoms can occur from contact of piglets with zearalenone and/or derivatives in the urine of the contaminated sows, even though in the majority of cases, the contamination occurs in gestation, through the sow’s feed, or after birth, via contaminated milk. Young boars are generally more affected than adults. The main toxicological clinical symptoms of these animals include growth of the mammary glands, edema of the foreskin that can hamper urination and cause testicular atrophy with impaired sperm quality and volume. A reduction in libido is frequently observed, even though, this aspect apparently does not interfere with the rate of conception when adult males naturally mount females.

70 The main effects are mostly related to reproductive animals, although growing and finishing pigs are also affected, reducing feed consumption by 100 to 150 g/day, causing significant losses in productivity. Other mycotoxins are produced under the same climatic condition ideal for the production of zearalenone. Therefore, feed refusal and vomiting are often attributed to the simultaneous occurrence of zearalenone and deoxynivalenol. Macroscopic Lesions The main lesions caused by zearalenone intoxication in swine are physiological alterations in the reproductive tract which are characterized by the accumulation of liquids, forming interstitial edema, proliferation of cells, and metaplasia of squamous epithelial cells of the vagina and cervix. The vulva, vagina, cervix, and uterus appear swollen because of the combination of edema with cell hypertrophia, and hyperplasia of the components of its structures (Figure 18). The uterotrophic effects of zearalenone bring about a significant enlargement of the uterus, particularly of the uterine horns. The ovaries of prepubertal females present and increase in volume with a large number of small follicles; however, there is no evidence of formation of the corpus luteum. Older females have large secondary follicle development and other follicles presented atresia. There is no evidence of ovulation because the ovaries did not present corpus luteum in the ovarian tissues of intoxicated female. Mammary glands and teats were edematous and exhibited a significant increase in size.

71 Figure 18 – Reproductive system of gilts showing alterations due to zearalenone intoxication. (A) Normal prepubertal gilt reproductive system. (B) Enlarged prepubertal gilt reproductive system after zearalenone intoxication 2 mg/kg of feed for 24 days.

A

B

Microscopic Lesions Histological alterations in the reproductive tract of swine, caused by zearalenone, include edema of the uterine wall, and metaplasia of the epithelium of the cervix and vagina. Histologically, hypoplasic ovaries and follicular atresia were observed, with a proliferation of connective tissue. The uterine walls, cervix, vagina, and vulva were all thickened (Figure 19), and edema and proliferation in all layers of these organs due to a combination of hyperplasia and hypertrophy was evident.
Figure 19 – Microscopic alterations caused by zearalenone in the mucosa of the reproductive

system of gilts. (A) Normal mucosa (control). (B) Thickened mucosa after intoxication with 2 mg of zearalenone/kg of feed for 24 days (Prof. Driemeier).

A

B

72 Diagnosis The manifestation of characteristic clinical symptoms of this intoxication occurs in approximately two weeks. Therefore, preventive measures, especially diagnosing contaminated feed before ingestion, are of fundamental importance. Frequently changing feed is an indicative factor in the occurrence of the illness because the initial clinical symptoms appear after 1 to 3 weeks of using a new batch of feed. Only macroscopically evaluating the raw material that makes up the animal’s diet does not guarantee that the feed will have been correctly analyzed and free of zearalenone. Sampling procedures, analysis and management of zearalenone are the same as those described in this book for other mycotoxins. Other than determining the presence of zearalenone in feed, plasma and feces can be analyzed. The extraction of zearalenone from feed is normally done with solvent combinations such as acetonitrile, ethyl acetate and methanol, which may be combined with water, depending on the type of matrix analyzed and the methodology used. The use of immunoaffinity columns for clarification of the samples and quantification using gas chromatography is an efficient method. Thin layer chromatography has been used often, and although some still value its use, it has been substituted by more efficient methods such as High Performance Liquid Chromatography (HPLC) combined with detecting ultraviolet or fluorescence. There are currently more sophisticated methods such as GC/ECD, GC/MS e HPLC/MS. ELISA methods are also used, but are not very sensitive, only quantifying one toxin at a time and are seriously deficient in repeating results. They are often used as semiquantitative methods. Treatment Clinical symptoms, such as edema of the vulva and perinatal mortality, disappear within 3 to 4 weeks when contaminated feed is substituted with feed free of zearalenone. Measurements to prevent the presence of this toxin in the feed should start with good agricultural and storage practices to avoid mold development. An efficient quality control system to evaluate contamination of feed ingredients can prevent the problem with relative

73 assurance. Redirecting zearalenone contaminated ingredients to more resistant species such as bovines and poultry, should be considered. The use of natural or modified adsorbents deserves further scientific study, since some have presented promising results in field situations. The use of dietary hepatic protectors such as methionine and choline has had good results, especially in restoring the appetite of intoxicated animals. Levels of this mycotoxin should not surpass 10 ?g/kg of feed, which in practice means that diets destined for swine should be free of zearalenone.

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74 BARTOS, J.; MATYAS, Z. The occurrence of zearalenone in domestic grains. Vet. Med. (Praha), v. 26, p. 505-512, 1981. BENNETT, G.A.; ANDERSON, R.A. Distribution of aflatoxin and/or zearalenone in wetmilled corn products: A review. J. Agric. Food. Chem., v. 26, p. 1055-1060, 1978. BIEHL, M.L.; PRELUSKY, D.B.; KORITZ, G.D.; HARTIN, K.E.; BUCK, W.B.; TRENHOLM, H.L. Biliary excretion and enterohepatic cycling of zearalenone in immature pigs. Toxicol. Appl. Pharmacol., v. 121, p. 152-159, 1993. BRANDENBERGER, A.W.; TEE, M.K.; LEE, J.Y.; CHAO, V.; JAFFE, R.B. Tissue distribution of estrogen receptors alpha (ER-alpha) and beta (ER-beta) mRNA in the midgestational human fetus. J. Clin. Endocrinol. Metab., v. 82, p. 3509-3512, 1997. BRANTON, S.L.; DEATON, J.W.; HAGLER W.M.; MASLIN, W.R.; HARDIN, J.M. Decreased egg production in commercial laying hens fed zearalenone-and deoxynivalenolcontaminated grain sorghum. Avian Dis., v. 33, p. 804-808, 1989. CHANG, W.M.; KUTTZ, J.H.; MIROCHA, C.J. Effects of the mycotoxin zearalenone on swine reproduction. Am. J. Vet. Res., v. 40, p. 1260-1267, 1979. DACASTO, M.; ROLANDO, P.; NACHTMAN, C.; CEPPA, L.; NEBBIA, C. Zearalenone mycotoxicosis in piglets suckling sows fed contaminated grain. Vet. Hum. Toxicol., v. 37, p. 359-361, 1995. DAILEY, R.E.; REESE, R.E.; BROUWER, A. Metabolism of [14C] zearalenone in laying hens. J. Agric. Food Chem., v. 28, p. 286-291, 1980. DALCERO, A.; MAGNOLI, C.; CHIACCHIERA, S.; PALACIOS, G.; REYNOSO, M. Mycoflora and incidence of aflatoxin B1, zearalenone and deoxynivalenol in poultry feeds in Argentina. Mycopathologia, v. 137, p. 179-184, 1997. DIEKMAN, M.A.; LONG, G.G. Blastocyst development on days 10 or 14 after consumption of zearalenone by sows on days 7-10 after breeding. Am. J. Vet. Res., v. 50, 1224-1227, 1989. EDWARDS, S.; CANTLEY, T.C.; ROTTINGHAUS, G.E.; OSWEILER, G.D.; DAY, B.N. The effects of zearalenone on reproduction in swine. I. The relationship between ingested zearalenone dose and anestrus in non-pregnant, sexually mature gilts. Theriogenology, v. 28, p. 43-49, 1987. EDWARDS, S.; CANTLEY, T.C.; ROTTINGHAUS, G.E.; OSWEILER, G.D.; DAY, B.N. The effects of zearalenone on reproduction in swine. II. The effect on puberty attainment and postweaning rebreeding performance. Theriogenology, v. 28, p. 51-58, 1987.

75 ETIENNE, M.; JEMMALI, M. Effets of the mycotoxin zearalenone on swine reproduction. Am. J. Vet. Res., v. 40, p. 1-10, 1982. FARNWORTH, E.R.; TRENHOLM, H.L. The effect of acute administration of the mycotoxin zearalenone to female pigs. J. Environ. Sci. Health B, v. 16, p. 239-252, 1981. FARNWORTH, E.R.; TRENHOLM, H.L. The metabolism of the mycotoxin zearalenone and its effects on the reproductive tracts of young male and female pigs. Can. J. Anim. Sci., v. 63, p. 967-975, 1983. FURLONG, E.B.; SOARES, L.M.; LASCA, C.C.; KOHARA, E.Y. Mycotoxins and fungi in wheat stored in elevators in the state of Rio Grande do Sul, Brazil. Food Addit. Contam., v. 12, p. 683-688, 1995. GAREIS, M.; BAUER, J.; THIEM, J.; PLANK, G.; GRABLEY, S.; GEDEK, B. Cleavage of zearalenone-glycoside, a 'masked' mycotoxin, during digestion in swine. J. Vet. Med. B, v. 37, p. 236-240, 1990. GROSS, V.J.; ROBB, J. Zearalenone production in barley. Ann. Appl. Biol., v. 80, p. 211-216, 1975. JAMES, L.J.; SMITH, T.K. Effect of dietary alfafa on zearalenone toxicity and metabolism in rats and swine. J. Anim. Sci., v. 55, p. 110-118, 1982. JEMMALI, M. Presence of an estrogenic factor of fungal origin: Zearalenone or F2, as a natural contaminant in corn. Ann. Microbiol. (Paris), v. 124, p. 109-114, 1973. JEMMALI, M.; MAZERAND, C. The presence of zearalenone or F2 in commercial walnuts. Ann. Microbiol. (Paris), v. 131b, p. 319-321, 1980. KALLELA, K.; SAASTAMOINEN, I. The effect of grain preservatives on the growth of the fungus Fusarium graminearum and on the quantity of zearalenone. Acta Vet. Scand., v. 22, p. 417-427, 1981. KALLELA, K.; SAASTAMOINEN, I. Decomposition of the Fusarium graminearum toxin zearalenone in storage conditions. Nord. Vet. Med., v. 33, p. 454-460, 1981. KOLLARCZIK, B.; GAREIS, M.; HANELT, M. In vitro transformation of the Fusarium mycotoxins deoxynivalenol and zearalenone by the normal gut microflora of pigs. Nat. Toxins, v. 2, p. 105-110, 1994. KORDIC, B.; PRIBICEVIC, S.; MUNTANOLA-CVETKOVIC, M.; NIKOLIC, P.; NIKOLIC, B. Experimental study of the effects of known quantities of zearalenone on swine reproduction. J. Environ. Pathol. Toxicol. Oncol., v. 11, p. 53-55, 1992.

76 KUIPER-GOODMAN, T.; SCOTT, P.M.; WATANABE, H. Risk assessment of the mycotoxin zearalenone. Regul. Toxicol. Pharmacol., v. 7, p. 253-306, 1987. KURTZ, H.J.; MIROCHA, C.J. Zearalenone (F-2) induced estrogenic syndrome in swine. In: SYLLIE, T.D.; MOREHOUSE, L.G., eds, Mycotoxic Fungi, Mycotoxins, Mycotoxicosis. An Encyclopedic Handbook, New York: Marcel Dekker, v. 2, p. 260-265, 1978. LONG, G.G.; DIEKMAN, M.A. Characterization of effects of zearalenone in swine during early pregnancy. Am. J. Vet. Res., v. 47, p. 184-187, 1986. LONG, G.G.; DIEKMAN, M.A. Effects of zearalenona on early gestation in gilts. J. Anim. Sci., v. 59, p. 1662-1670, 1984. LONG, G.G.; DIEKMAN, M.A.; TUITE, J.F.; SHANNON, G.M.; VESONDER, R.F. Effect of Fusarium roseum corn culture containing zearalenone on early pregnancy in swine. Am. J. Vet. Res., v. 43, p. 1599-1603, 1982. LONG, G.G.; DIEKMAN, M.A.; SCHEIDT, A.B. Effect of zearalenone on days 7 to 10 postmating on intrauterine environment in sows and migration of embryos in sows. J. Anim. Sci., v. 66, p. 452-458, 1988. LONG, G.G.; TUREK, J., DIEKMAN, M.A.; SCHEIDT, A.B. Effect of zearalenone on days 7 to 10 post-mating blastocyst development and endometrial morphology in sows. Vet. Pathol., v. 29, p. 60-67, 1992. LONCOREVIC, A.; JOVANOVIC, M.; LJESEVIC, Z.; STANKOV, Z.; BOGETIC, V.; TOSEVSKI, J. Appearance of vulvovaginitis in new-born piglets originated from sows fed diet contaminated with Fusarium graminearum. Acta Vet. Yugoslavia, v. 27, p. 151-157, 1977. MILLER, J.K.; HACKING, A.; HARRISON, J.; GROSS, V.J. Stillbirths, neonatal mortality and small litters in pigs associated with the ingestion of Fusarium toxin by pregnant sows. Vet. Rec., v. 93, p. 555-559, 1973. OLSEN, M.; MALMLOF, K.; PETTERSSON, H.; GRAJEWSKI, J. Influence of dietary fiber on plasma and urinary levels of zearalenona and metabolites in swine. Mycotoxin Res., v. 7, p. 8-11, 1991. PALYUSIK, M.; HARRACH, B.; MIROCHA, C.J.; PATHRE, S.V. Transmission of zearalenone and zearalenol into porcine milk. Acta Vet. Acad. Sci. Hung., v. 28, p. 217222, 1980.

77 PALYUSIK, M.; HARRACH, B.; HORVATH, G.; MIROCHA, C.J. Experimental fusariotoxicosis of swine produced by zearalenone and T-2 toxins. J. Environ. Pathol. Toxicol. Oncol., v. 10, p. 52-55. 1990. PESTKA, J.J., TAI.; J.H., WITT, M.F.; DIXON, D.E.; FORSELL, J.H. Suppression of immune response in the B6C3F1 mouse after dietary exposure to the Fusarium mycotoxins deoxynivalenol (vomitoxin) and zearalenone. Food Chem. Toxicol., v. 25, p. 297-304, 1987. PULLAR, E.M.; LEREW, W.M. Vulvovaginitis of swine. Aust. Vet. J., v. 13, p. 28-31, 1937. RUHR, L.P.; OSWEILER, G.D.; FOLEY, C.W. Effect of the estrogenic mycotoxin zearalenone on reproductive potential in the boar. Am. J. Vet. Res., v. 44, p. 483-485, 1983. SABINO, M.; PRADO, G.; INOMATA, E.I.; PEDROSO, M.; GARCIA, R.V. Natural occurrence of aflatoxins and zearalenone in maize in Brazil. Part II. Food Addit. Contam., v. 6, p. 327-331, 1989. SHREEVE, B.J.; PATTERSON, D.S.P.; ROBERTS, B.A.; WRATHALL, A.E. Effect of moldy feed containing zearalenone on pregnant sows. Br. Vet. J., v. 134, p. 421-427, 1978. VANYI, A.; SZEKY, A. Fusariotoxicosis. 6. The effect of F-2 toxin (zearalenone) on the spermatogenesis of male swine. Magy Allattorv. Lapja, v. 35, p. 242-246; 1980. YOUNG, L.G.; KING, G.J. Zearalenone and swine reproduction. J. Am. Vet. Med. Assoc., v. 185, p. 334-335, 1984. YOUNG, L.G.; KING, G.J. Low concentrations zearalenona in diets of boars for a prolonged period of time. J. Anim. Sci., v. 63, p. 1197-2000, 1986. YOUNG, L.G.; VESONDER, R.F.; FUNNEL, H.S.; SIMONS, L.; WILCOCK, B. Moldy corn diets of swine. J. anim. Sci., v. 52, p. 1312-1318, 1981. YOUNG, L.G.; HE, P.; KING, G.J. Effects of feeding zearalenona to sows on rebreeding and pregnancy. J. Anim. Sci., v. 1, p. 15-20, 1990.

78

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FUMONISINS

79 FUMONISINS Summary Fumonisin are produced by the secondary metabolism of various toxigenic fungi of the Fusarium and Alternaria genera, with Fusarium moniliforme recognized as the largest producer of this mycotoxin. Approximately two dozen fumonisins are known. However, only fumonisins B1 (FB1), B2 (FB2) and B3 (FB3) are toxicologically significant. They occur in a variety of grains, especially corn, where they display concentrations that generally induce subclinical intoxications in different species. These toxins inhibit the acetyl transferase enzyme, which is responsible for the transformation of sphingolipids. Consequently, there is a buildup of sphinganine and sphingosine in the blood stream. Equine are the most sensitive of domestic species to FB1, manifesting clinical symptoms that include injury to the nervous system and leukocephalomalacia (ELEM). Swine are also highly sensitive to fumonisins, which can cause pulmonary edemas, among other clinical symptoms. The characteristic clinical symptoms of fumonisin toxicosis in swine include difficulty breathing and cyanosis due to pulmonary edema. Chronic intoxications are the most frequent and it is characterized by a decrease in productivity. Other than producing lesions, characteristic in this species, the liver is affected by fumonisin toxicosis outbreaks. The clinical incidence of pulmonary edema caused by fumonisins has not been greatly studied in Brazil. History Fumonisin was first identified in 1988, therefore, its toxic effect on animals has been known for many years. Different outbreaks of equine leukocephalomalacia have been attributed to the ingestion of contaminated feed. In 1902 the illness was observed in North American equine fed with corn which was highly contaminated with F. moniliforme. The intoxicated animals presented characteristic clinical symptoms including neurological dysfunction and corticocerebellar lesions. From 1934 to 1935 close to five thousand equine deaths were recorded in Illinois and Iowa, USA. Clinical symptoms and lesions were identical to those attributed to leukoencephalomalacia in previous outbreaks. Different organisms were found in the feed consumed by the affected animals, among those found were Swine fumonisin toxicosis: difficulty breathing, cyanosis, and pulmonary edema.

80 fungi from the Fusarium genus. Cultures of Fusarium moniliforme in sterile food were used to reproduce the disease, which confirmed the toxigenic potential of the fungus and its role in the production of certain toxins. In Brazil, the illness was first described and accounted for in the state of S?o Paulo in 1950. Beginning in 1982, hundreds of outbreaks have been reported in Brazil. Scientists believe that the disease appears to be more important than indicated by the few reports available. Mistakes in diagnosing the illness in cases that did not result in death and where feed was not monitored could have distorted the perceived prevalence of the disease. Etiology In 1988 the structural formula of fumonisin B1 (FB1) molecule was discovered, but the natural occurring mycotoxin was first isolated in 1990. Dozens of fumonisin molecular structures have been isolated, among them: FB1, B2, B3, B4, B5, A1, A2, A3, AK1, BK1, C1, C3, C4, P1, P2, P3, PH1a and PH1b. The predominant mycotoxin produced by strains of Fusarium moniliforme is FB1, a diester of propane 1, 2, 3-trycarboxylic acid and 2-amino-12, 16 dimetil-3, 5, 10, 14, 15-pentahydroxycosan, where carbons 14 and 15 of the hydroxyl groups are esterified with carboxyterminal propane 1, 2, 3-trycarboxylic acid group (Figure 20) Figure 20 – Molecular structures of some fumonisins (Norred, 1993). CH3 O O=C HO2C-HC HO2C O C=O R1 FB1 FB2 FB3 FB4 FA1 FA2 H OH H OH H OH H OH OH OH OH H R2 OH OH OH H CH3CO OH OH R3 H H H R4 CH-CO2H CO2H CH3 R1 R2 R3 NHR4

CH3-(CH2)3-CH-CH-CH-CH2-CH-CH2-CH-(CH2)4-CH-CH2-CH-CH-CH3

H2C CH2 H2C CH2

OH CH3CO

81 Fumonisins are highly polar molecules, soluble in water. They are more soluble in acetonitrile/water or methanol and insoluble in organic solvents. FB1 is very resistant to high temperatures, exhibiting few losses in the drying and processing of grains. Half (50%) of FB1 is recuperated when it is submitted to 150°C, 125°C, 100°C and 75°C for 10, 38, 75 minutes, and 8 hours, respectively. Other researchers have tested the stability of FB1 and FB2 for 60 minutes at 150°C in media containing different potential of hydrogen. There was a 80 to 90% loss of toxins at pH levels of 4.0; 18 to 30% at pH levels of 7.0, and 40 to 52% at pH levels of 10.0. Occurrence of fumonisins in feed and food Fumonisins are mycotoxins, secondary metabolites of fungi, especially of the Fusarium genus. Fumonisins are produced mainly by F. moniliforme, but other species such as F. proliferatum, F. nygamai, F. anthophilum, F. dlamini, and F. napiforme are also producers. It has been recently shown that fungi of the Alternaria spp genus also produce fumonisin, but in smaller quantities. Fungi of the Fusarium genus develop and produce the largest quantities of fumonisins in climatic conditions with temperatures between 15 and 25°C and high contents of substrate humidity, above 20% or water activity higher than 0.9. Fungi of the Fusarium genus are often called “field fungi” because the largest concentrations of toxins are seen in grains that are exposed during tillage to these basic conditions for fungal development. The predominant flora of different grains which are freshly harvested, generally contain fungi of the Fusarium genus. However, it is known that the incidence of fumonisins is significantly higher in cereals that are exposed to bad weather during and after the maturation of the grains. This generally occurs when grains are not harvested or stored immediately after physiological maturation. Fumonisins are found globally, especially FB1, which is responsible for approximately 70% of all quantified fumonisins in cereals and their byproducts. FB2 and FB3 occur jointly in smaller proportions. Normally, only FB1, FB2 e FB3 are detected when fumonisins are produced under natural conditions. Numerous researchers have proposed that fumonisins C1, FB4, FA1, and FA2 can only be produced in laboratory conditions. However, apart from FB1, FB2, and FB3 some researchers have found other fumonisins such as FB4, C1, High humidity + mild temperatures = production of fumonisins. Thermal processing of foods does not affect fumonisins.

82 C3 and C4 (which had only been isolated in laboratory conditions) in corn samples which were sufficiently damaged. Agricultural products frequently contaminated with fumonisins include: oatmeal, barley, bran, rice, corn, and their derivatives. The presence of FB1 at concentrations between 44 and 83 mg/kg is very frequent in corn coming from the Republic of Transkei in Southern Africa, a region with a high incidence of human esophageal cancer, but even higher concentrations of up to 117 mg of FB1/kg have been found in corn produced in this region. The incidence of high concentrations of fumonisins in human food in Transkei, Italy, and China was epidemiologically compared to the incidence of esophageal cancer in the population. This comparison showed that there is a highly significant index of correlation between the two. Concentrations of up to 7.2 and 8.85 mg of FB1/kg of corn and its derivatives were found in the USA and South Africa, respectively. FB1 was detected at levels between 0.2 mg/kg and 330 mg/kg in feed involved in episodes of intoxication in different animal species (such as horses and swine). In Europe, analyses to detect naturally occurring FB1 and FB2 performed on corn samples from various countries, demonstrated that batches of cereals frequently present 100% positivity. Although samples have demonstrated a high rate of 50 to 90% of corn from any given region can be contaminated with fumonisins.

positivity, concentrations normally found are low, varying from 0.055 and 5.0 mg/kg of feed. In South America molds of the Fusarium genus also find favorable conditions for development and production of fumonisins. In surveys conducted in Uruguay on 64 samples of feed, 32 samples (50%) were positive for FB1, with contamination varying from 0.005 up to 6.34 mg/kg. In the province of Cordoba, Argentina, 50 hybrids of corn analyzed presented 100% positivity, with concentrations ranging from 0.185 up to 27.05 mg/kg of FB1 and 0.04 up to 9.95 mg/kg of FB2. In Brazil, the presence of fumonisins in cereals and feed was found by various researchers. The mycotoxin has greater occurrence in the Southern states of the country, but it is also present in the Southeast, Goias, and Mato Grosso do Sul. The most frequently monitored products are corn, cornmeal, and feed for poultry and swine. The incidence of fumonisins has been found in most of the samples that have been analyzed, presenting 50 to

83 90% positivity. Concentrations of fumonisins are generally low, between 0.005 and 15 mg/kg of samples, with an average of less than 1 mg/kg of feed. Nonetheless, samples containing concentrations higher than 50 mg of FB1/kg of feed were frequently found. In the state of Rio Grande do Sul, 407 samples (267 samples of corn, 92 samples of feed, 8 samples of oatmeal, 5 samples of rice bran, 8 samples of soybean meal, 14 samples of barley, and 13 sample of wheat) obtained from different storage facilities and feed mills from the Southern region of Brazil were analyzed during the period between 1996 and June 1998. Of the samples analyzed, 32.2% were positive for FB1, with levels of contamination varying from 86 ?g/kg to 78,9 mg/kg. The highest concentration found and the percentage of positive samples were 14.2 mg/kg and 75%, respectively in rice bran; 68.3 mg/kg and 30.4% in feed; 78.9 mg/kg and 35.2% in corn; 2.4 mg/kg and 7.7% in wheat; 175.5 ?g/kg and 25% in oatmeal; 2.4 mg/kg and 14.3% in barley. Soybean meal did not present contamination levels above the quantification limit of the method used (50 ?g/kg). In evaluations done in the last 17 years on samples from the main grain producing states of Brazil, LAMIC/UFSM analyzed fumonisins in 45,558 samples, finding an average concentration of FB1 + FB2 of 1591.1 ?g/kg. Corn represented 22,746 of these samples, with an average contamination of 1,989.7 ?g/kg, found in 75% of this grain. Feed submitted for fumonisin analysis presented 72% contamination of all samples with an average level of 1,592 ?g/kg.. Toxicokinetics of fumonisins Absorption The bioavailability of FB1, when administered orally, is relatively low in various species of domestic animals. The absorption of the mycotoxin ingested by swine was around 3 and 6%. Due to the low absorption of FB1 in the gastrointestinal tract, high concentrations are eliminated in feces. Elimination through urine is low, despite the fact that parenteral administration could have invert quantities eliminated through these two ways. Approximately 30 to 45 minutes after intragastric administration in pigs, the toxin could be detected in plasma, with maximum concentrations appearing between 60 and 90 minutes, reaching no detectable levels in a few hours.

84 Distribution The distribution of radioactivity in the tissue of pigs fed with a diet containing FB1 radioactive labeled by 14C was tested using 3 mg of toxin/kg for 12 days and 2 mg of FB1/kg until completing 24 days of intoxication. At day 25 these animals were fed uncontaminated rations for 9 days. Samples from different organs were collected on days 3, 6, 12, 24, 27, and 33 of the experiment. Radioactivity was detected from the first sampling, with levels of concentrations in the tissues increasing until day 24. The highest concentrations in tissue were found in the liver and kidneys (160 and 65 ?g/kg, respectively). After 3 days of consuming uncontaminated feed, radioactive labeled residues in the organs decreased by close to 35%. After the 6th day of ingesting a fumonisin free diet, only traces of radioactivity were detected. At the end of the experiment, no residue was detected in the plasma, spleen, muscle, brain, adrenal glands, fat, or skin. Elimination The elimination of fumonisin from the plasma is fast in all species. Swine, particularly, present a relatively long terminal elimination phase (γ) of t? + 182.6 minutes, and the plasmic concentration of toxin is less than the detectable limit (18 ?g/kg) 4 hours after administration, when the purified toxin is administered orally in moderate doses. It is also known that the elimination of fumonisins occurs for a longer period when naturally contaminated feed is ingested. Recovery from the toxin is higher than 90%, mostly eliminated through feces and only 0.6% through urine. Based on the evaluation of concentrations, enterohepatic circulation, and elimination, it has been concluded that the bioavailability of FB1 is around 4%. Residues Fumonisins are quickly eliminated from the organism of the intoxicated animals, especially when contaminated feed is substituted by feed not containing the toxin. Poultry and bovines are very resistant to fumonisins, eliminating the toxin through eggs and milk when consuming feed with high concentrations of these mycotoxins. During the period of intoxication, animal hair incorporates the toxin in the area of growth. This finding demostrates the possibility of stipulating the periods when the animal was intoxicated by quantifying the toxin from different sections of animal hair strands.

85 Pathogenesis The molecular structures of fumonisins are similar to those of sphinganine and sphingosine, which are responsible for the formation of sphingolipids (sphingomieline and glycosphingolipids - Figure 21) in animals. More than three hundred sphingolipids are synthesized in the endoplasmatic reticulum of eukaryotic cells from a combination of serine with palmitoyl-CoA. They are very important in maintaining the integrity of the cellular membrane, regulating surface cellular receptors, ion pumps, and other systems that are vital to the functioning and survival of the cell. The glycosphingolipid galactosylceramide lipid is the main constituent of myelin, which is a component of oligodendrocytes membrane and Schwann cells in central and peripheral nervous system, respectively. Studies done on rodent hepatocytes have demonstrated that fumonisins block the formation of sphingolipids by inhibiting the enzyme N-acyltransferase. Although the mechanism of action is not completely understood, an outline representing the mechanism has been proposed based on the similarity between fumonisins and sphingolipid molecules (Figure 22). The accumulation of sphinganine and, sometimes sphingosine, has been demonstrated in vitro using cells from the intestinal epithelium of swine, and in vivo with the intoxication of ponies, pigs, and chickens. There was partial or total inhibition of sphingosine and of enzyme N-acyltransferase was due to hepatotoxicosis in pigs. Some researchers have proposed that there could also be an activation of intravascular macrophages in the lungs of pigs. The presence of those phagocytic cells adhere to the endothelium of pulmonary capillaries, with subsequent release of vasoactive substance, could be the cause of pulmonary lesion susceptibility of pigs intoxicated with fumonisins. Accumulation of membrane material was also detected in the periphery of cellular nuclei, components of the vascular endothelium of pig lungs that consume fumonisins. This alteration was not found in endothelial cells of other organs or in lungs of other species, leading researchers to conclude that this alteration in the lungs could be a result of an increase in capillary permeability which induces pulmonary edema in intoxicated pigs.

86 Other researchers have found a series of alterations in cardiac dynamics and suggested that the formation of pulmonary edema in swine could be caused by contractile deficiency of The pathogenesis of fumonisins in swine suggests cardiotoxic action.

the left side of the heart and not by increased capillary permeability of the lungs. Figure 21 – Chemical structure of fumonisins B1, B2, sphinganine and sphingosine.

OR

OH

OH CH3

Fumonisina B1 Fumonisin B1

CH3

OR

CH3

OH OH OH

+

NH3

OR

CH3 CH3 OR CH3 OH CH2 OH
+ +

Fumonisin B2 2

Fumonisina B

N H3

Esfinganina Sphinganine

N H3

OH CH2 OH
+

Sphingosine Esfingosina

NH3

O R= HOOC COOH

Wang et al., 1991

Fumonisins can exert carcinogenic activity on rat hepatocytes when exposed to high doses of the toxin. These toxins are mitogenic in fibroblasts of mice, stimulating cellular multiplication due to the increase in sphingolipid concentration, as well as products of its metabolism. They induce apoptosis in intoxicated animals and in cell cultures, through mechanisms that are still unclear.

87 The incidence of fumonisins in food destined for human consumption has been epidemiologically related to incidences of esophageal cancer since 1976 in Transkei, Italy, and China. Based on toxicological evidence, the International Agency for Research on Cancer (IARC) has declared that toxins produced by Fusarium moniliforme are possible human carcinogens (class 2B carcinogen). Figure 22 – Mechanism of sphingolipid formation inhibition by fumonisin.

Palmitoyl-CoA + Serina 3-Ceto sphinganine ← 3-CETOSPHINGANINE REDUCTASE
N-ACYL + TRANSFERASE

Sphinganine

Acyl-CoA CoA Dihydroceramide
N-ACYLTRANSFERASE



SPHINGANINE REDUCTASE

Ceramide (sphingosine + fatty acid)
Choline Phosphate one or more sugar resídues

Sphingosine CoA Acyl-CoA

Sphingomieline

Glycosphingolipids
( return)

X = sphingolipid formation inhibition by fumonisin Wang et al.,1991

88 Immunosuppressant effects of fumonisins on swine The immunopathogenic potential of fumonisins preoccupies the animal industry, even though few studies show the real importance of fumonisins in the decrease of intoxicated animal immunological capacities. However, statistical studies confirm the involvement of FB1 in the occurrence of Mystery Swine Disease (known today as PRRS, caused by the virus– PRRSv) in the United States. Researchers have concluded that the association of fumonisin toxicity and PRRS were significant in 21 herds studied. Of the 12 that presented clinical signs of the disease, 8 consumed feed containing more than 20 mg/kg of FB1, and of the 9 herds that were not affected by the disease, only 1 consumed feed containing more than 20 mg of FB1/kg. Decrease in the concentration of macrophages in the lung was found beginning with 1 week of intoxication in piglets fed a diet containing 20 mg of FB1/kg. Associated with this decrease in immunity, a greater susceptibility to infection by Pseudomonas aeruginosa was seen. The increase in susceptibility to Escherichia coli in intoxicated piglets has also been proven. Doses of 0.5 mg of FB1/kg of live weight/day during 7 days were sufficient to significantly increase the bacterial dissemination when Escherichia coli was administered orally. Although animals did not develop lesions derived from the intoxication by FB1, researchers concluded that bacterial colonization, after 24 Pneumopathies could be associated to the ingestion of fumonisins.

hours, was significantly higher in the lungs, spleen, kidneys, and greater still in the digestive organs such as the ileum, cecum, colon, and mesenteric lymph nodes. It was also observed that animals intoxicated with FB1 presented fewer number of local inflammatory cytokines. Clinical symptoms Acute intoxication of swine with fumonisins induces clinical symptoms characterized by pulmonary edema, which generally occurs between 3 and 5 days after the initial consumption of contaminated feed. In these cases, death can occur within a few hours. Chronic intoxication occurs as a result of consuming feed with low concentrations of the toxin for a prolonged period of time, causing hepatic damage in affected pigs. Respiratory problems could be associated with fumonisin intoxication.

Concentrations of FB1 higher than 10 mg/kg in feed are considered unsafe for swine. However, natural outbreaks of pulmonary edema have been found in swine fed diets contaminated with 1 mg of FB1/kg (Table 13). Various researchers found that swine at

89 reproductive age are more sensitive to intoxication by fumonisins when compared to growing piglets. However, stress factors, such as nutritional and management deficiencies could also be important factors in the onset of intoxication. Females are less sensitive than males, but gestating females that are intoxicated generally have offspring with pulmonary lesions characteristic of pulmonary edema. Levels of fumonisins were not detected in the milk of sows that were fed rations containing concentrations of 100 mg FB1/kg of feed and nursing piglets did not present any signs of intoxication. Abortions resulting from intoxication by fumonisins generally did not cause complications in subsequent gestations. The principal clinical symptoms in swine chronically intoxicated by fumonisins are nonspecific and occur as a consequence of hepatic lesions, leading to an overall sickly appearance of the animal, and a decrease in weight gain. These symptoms can be easily confused with malnutrition, genetic deficiency, inadequate management and/or other afflictions that also cause clinically sickly appearance of the animal. The herd generally presents a significant increase in non-uniformity. There is usually a decrease in feed intake and weight gain, which can often lead to significant worsening of food conversion. Bristles present a decrease in shine, coarseness, and animals roam more frequently through the pen without eating. Acute intoxication of swine by fumonisins is clinically easier to detect because it is characterized by pulmonary edema. The most characteristic signs seen in affected animals include anorexia, lethargy, open mouth, increase in respiratory frequency, jaundice, and cyanosis of the skin, most evident on the ears, snout, sclera and mucosa membranes. Excessive salivation can also occur, as well as hepatic encephalopathy syndrome, a humid snoring sound when listening to the lungs, and watery diarrhea. After the decreased in intake induced by the contaminated feed, respiratory difficulties, comprised of superficial and abdominal breathing associated with an increase in frequency, normally occur between 12 and 24 hours. Characteristic symptoms of pulmonary edema appear close to 3 to 5 days after consumption of contaminated feed. If toxicosis persists, more severe symptoms such as ataxia of the posterior limbs, dog sitting position, lateral decubitus, a short period of agony, and death are rapidly observed (Figure 23).

90 Figure 23 – Sequence of clinical symptoms in pigs with pulmonary edema induced by intoxication with fumonisins. (A) Apathy and prostration on the onset of clinical symptoms of pulmonary edema. (B) Dog sitting position in order to relieve pulmonary pressure. (C) Cyanosis of extremities and intensification of apathy. (D) Increase in respiratory difficulties and initiation of oral respiration. (E) Death of animal due to intense pulmonary edema. A B

C

D

E

Hemoptysis and bloody feces can be present in some animals of the intoxicated herd. Generally more than 80% of the animals of the same lot are affected. When intoxication is not severe and contaminated feed is immediately replaced, the recuperation rate of the lot can reach up to 100%. However, if intoxication is very severe, all affected animals can die within a few hours.

91

Table 13 – Maximum recommended levels of total fumonisins (FB1 + FB2 + FB3) in corn, corn-based feed, and total feed of various species. Animal Species Maximum limits of total fumonisins in corn and its byproducts (mg/kg1) 5 5 20 10
4

Correlation Factor2

Maximum limits of total fumonisins in feed (mg/kg1)

Equine3 Rodents Unscaled fish Swine Ruminants Mustelids5 Chicken6 Ruminants and laying Hens7 All others8
1

0.2 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5

1 1 10 10 30 30 50 15 5

60 60 100 30 10

http://www.cfsan.fda.gov Total fumonisins = FB1 + FB2 + FB3. 2 Portion of corn or corn mixed with other feed. 3 Includes donkeys, zebras, and other wild equine. 4 Includes cattle, sheep, goats, and other ruminants for human consumption, older than 3 months old. 5 Wild animals bred for fur production. 6 Turkeys, hens, ducks, and other poultry for human consumption. 7 Includes laying poultry, roosters, milking cows, bulls, and buffalo. 8 Includes canines and felines.

Clinical Pathology Determining the clinical biochemical parameters is of great importance in the evaluation of the extension and prognosis of the pathology. Generally alterations in biochemical concentrations are indicative of progression of the pathology of a given organ such as the liver, kidneys, or pancreas. Swine intoxicated with fumonisins present a Pulmonary edema ? Remember fumonisins.

significant increase in bilirubin, and in enzyme activity (aspartate aminotrasferase and glutamyltransferase), and above all, in alkaline phosphatase. Increased levels of serum

92 cholesterol generally occur on the 10th day of intoxication, and enzyme alterations occur even earlier. An increase in sphinganine and/or sphingosine, or an increase in the proportional concentration of these sphingolipids, has been considered to be a biological marker of intoxication with fumonisins. It is currently known that other mycotoxins also interfere with the concentrations of these two substances. Hematological parameters such as erythrocytes/mm3, hematocrit levels, hemoglobin concentrations, absolute hematometric indices, and leukogram are not easily affected by fumonisin-toxicosis in pigs. Total protein concentrations and albumine can, however, be affected, especially after severe hepatopathies. Macroscopic Lesions The necropsy of pigs intoxicated with high doses of fumonisins showed extensive pulmonary edema which frequently evolved into hydrothorax, jaundice, and darkening and hardening of the liver. Exposure to low doses during prolonged periods induced hepatic degradation and necrosis, interfering with protein synthesis, weight gain, and general animal performance (Figure 24). Figure 24 – Hepatic damage in pigs intoxicated with 30 mg of fumonisins/kg of feed for 21 days. (A) Livers of pigs not intoxicated with fumonisins. (B) Livers of intoxicated pigs presenting yellow coloration. A B

93

Necrosis of the medullary region, resulting from fumonisin intoxication is normally associated with hepatosis. Pathological changes observed in pigs affected by pulmonary edema are characterized by the presence of beige-clear or yellowish-gold transudate in the thoracic cavity, which characterizes more severe cases of pulmonary edema and hydrothorax. Concentrations greater than 500 mL of this yellowish-gold liquid can accumulate, initiating the coagulation process when it comes into contact with air (Figure 25). Figure 25 – Occurrence of pulmonary edema in pigs intoxicated for a period of 21 days with 30 mg of fumonisins/kg of feed. (A) Liquid from edema (arrows). (B) Lung of non-intoxicated swine. (C) Enlarged lung of intoxicated swine.

A

B

C

The lungs of pigs affected by pulmonary edema did not collapse when removed from the chest, and they presented an increase in size with rounded edges. The lungs are heavier because of the concentration of liquid in the parenchyma and air conduits. Edema predominantly occurs in the interlobular and interstitial region. When the accumulation is interlobular, it predominantly appears in the hilum, extending through the lung, separating the lobules by 3-5mm spaces. Exudate seeping through the pulmonary surface was observed, as well as distended septa and removal of the pleura, separating it from the pulmonary parenchyma. Little liquid is normally found in the bronchioles, bronchi and trachea.

94 Few pathological changes can be detected in different organs of pigs affected with pulmonary edema. However, some researchers assure that ventricular walls of the heart can become more flaccid. The incidence of ulcerative lesions in the esophagus and stomach, sometimes associated with the formation of hepatic and esophageal hyperplasia, can be observed in some chronically intoxicated animals. An increase of mediastinal and peribronchial lymph nodes was also observed in some animals. Microscopic Lesions Microscopic pulmonary lesions of pigs affected with pulmonary edema are similar, including an accumulation of acidophilic fibrin in the alveoli and interlobular lymphatic vessels. This protein rich fluid is present in abundance in the subpleural lymphatic vessels and has little cellular infiltration. The septum presents a small number of mononuclear cells and neutrophils. Some alveoli have few mononucleous cells and large amounts of capillary hyaline thrombi are found in the lungs. In cases of acute pulmonary edema, lesions in other organs are rarely found, but, hepatopathies with foci of necrosis can occur. Hepatic changes, insufficient to cause clinical pulmonary edema, are observed with greater frequency in animals that suffer chronic intoxication with low doses. Random hepatocellular necrosis, nuclear pleomorphism, an increase in the amount of mitosis, deformed hepatomegalocytosis cells and focal necrosis of hepatocytes can occur. Diagnostic, treatment, and prophylaxis Diagnosing acute swine fumonisin-toxicosis is easily done by identifying clinical symptoms. However, a definitive diagnosis is based on both the presence of lesions and analysis of the presence of fumonisins in feed. Various analytical methods such as Thin Layer Chromatography, Gas Observation of clinical symptoms leads to diagnosis.

Chromatography, Gas Spectrometry, ELISA,

Capillary Electrophoresis, and High

Performance Liquid Chromatography (HPLC) have been developed to quantify the amount of fumonisins in feed. Animals intoxicated by fumonisins, can also have an increase in sphinganine and/or sphingosine concentration in their blood stream, which can sometimes be used as biological markers of intoxication with fumonisins.

95 Intoxication of pigs with fumonisins does not have a specific treatment. The clinical symptoms of acute intoxication disappear approximately 3 days after withdrawing the contaminated feed. Prophylaxis consists of implementing measures that lead to the inhibition of fungal development in grains and ingredients used in feed, and/or a system which monitors the presence of mycotoxins in feed. BIBLIOGRAPHY BINKERD, K.A.; SCOTT, D.H.; EVERSON, R.J.; SULIVAN, J.M.; ROBINSON, F.R. Fumonisin of the 1991 Indiana corn crop and its effects on horses J. Vet. Diagn. Invest., v. 5, p. 653-55, 1993. COLVIN, B.M.; COOLEY, A.J.; REAVER, R.W. Fumonisin toxicoses in swine: Clinical and pathologic findings. J. Vet. Diagn. Invest., v. 5, p. 232-41, 1993. DIAS, S.M.C.; MALLOZZI, M.A.B.; CORR?A, B.; ISRAEL, W.M.; GON?ALEZ, E. Fluorimetric quantitation of opa-derivatives of fumonisins B1 and B2 in corn and Fusarium moniliforme culture extracts. Arq. Inst. Biol., S?o Paulo, v. 66, p. 69-75, 1999. DILKIN, P., MALLMANN, C.A.; ALMEIDA, C.A.A.; CORR?A, B. Robotic automated clean-up for detection of fumonisins B1 and B2 in corn and corn-based feed by highperformance liquid chromatography. J. Chromat. A, v. 925, p. 151 - 7, 2001. DILKIN, P. Micotoxicose suína: aspectos preventivos, clínicos e patológicos. Biológico, S?o Paulo, v. 64, n. 2, p. 187-91, 2002. DILKIN, P., MALLMANN, C.A.; ALMEIDA, C.A.A.; STEFANON, E.B.; FONTANA, F.Z.; MILBRADT, E.L. Production of fumonisins by strains of Fusarium moniliforme by varying conditions of temperature, moisture and growth period. Braz. J. Microb. v. 33, p. 111-8, 2002. DILKIN, P.; HASSEGAWA, R.; REIS, T.A.; MALLMANN, C.A.; CORR?A, B. Intoxica??o experimental de suínos por fumonisinas. Ciência Rural, v. 34, n. 3, p. 175-81, 2004. DILKIN, P.; ZORZETE, P.; MALLMANN, C.A.; GOMES, J.D.F.; UTIYAMA, L.L.; OETTING, L.L.; CORR?A, B. Toxicological effects of chronic low doses of aflatoxin B1 and fumonisin B1-containing Fusarium moniliforme culture material in weaned piglets. Food Chem Toxicol., v. 41, p. 1345-53, 2003.

96 DUPUY, J.; BARS, P.; BOUDRA, H. Termoestability of fumonisin B1 a mycotoxin from Fusarium moniliforme, in corn. Appl. Environ. Microbiol., v. 59, p. 2864-7, 1993. HASCHEK, W.M.; MOTELIN, G.; NESS, D.K.; HARLIN, K.S.; HALL, W.F.; VESONDER, R.F.; PETERSON, R.E.; BEASLEY, V.R. Characterization of fumonisin toxicity in orally and intravenously dosed swine. Mycopathologia, v. 117, p. 83-96, 1992. HIROOKA, E.Y.; YAMAGUCHI, M.M.; AOYAMA, S.; SUGIURA, Y.; UENO, Y. The natural occurrence of fumonisins in Brazilian corn kernels. Food Addit. Contam., v. 13, p. 173-83. 1996. http://www.cfsan.fda.gov/~dms/fumongu2.html acesso em 21 de novembro de 2006. http://www.lamic.ufsm.br/, extraído em 13 de agosto de 2004. LEESON, S.; GONZALO, J.D.G.; SUMMERS, J.D. Poultry disorders and mycotoxins. Guelph, Ontario, Canada, p. 249- 98, 1995. MALLMANN, C.A.; SANTURIO J.M.; DILKIN P. Equine leukoencephalomalacia associated with ingestion of corn contaminated with fumonisin B1. J. Braz. Soc. Microbiol., v. 30, p. 249-52, 1999. MALLMANN, C.A.; SANTURIO, J.M.; ALMEIDA, C.A.A.; DILKIN P. Fumonisin B1 levels in cereals and feeds from southern Brazil. Arq. Inst. Biol., v. 68, p. 41-5, 2001. MOTELIN, G.K.; HASCHEK, W.M.; NESS, D.K.; HALL, W.F.; HARLIN, K.S. SCHAEFFER, D.J.; BEASLEY, V.R. Temporal and dose-response features in swine feed corn screenings contaminated with fumonisin mycotoxins. Mycopathologia, v. 126: 27-40, 1994. OSWEILER, G.D.; ROSS, P.F.; WILSON, T.M.; NELSON, P.E.; WITTE, S.T.; CARSON, T.L.; RICE, L.G.; NELSON, H.A. Characterization of an epizootic of pulmonary edema in swine associated with fumonisin in corn screenings. J. Vet. Diagn. Invest., v 4, p. 53-9, 1992. PRELUSKY, D.B.; TRENHOLM, H.L.; SAVARD, M.E. Pharmacokinetic fate of labelled fumonisin B1 in swine. Nat. Toxins., v. 2, p. 73-80, 1994. RAMIREZ, M.L.; PASCALE, M.; SCHULZE, S.; REYNOSO, M.M.; MARCH, G.; VISCONTI, A. Natural occurrence of fumonisins and their correlation to Fusarium contamination in commercial corn hybrids growth in Argentina. Mycopathologia, v. 135, p. 29-34, 1996.
14

C-

97 ROSS, P.F.; RICE, L.G.; PLATTER, R.D.; OSWEILER, G.D.; WILSON, T.M.; OWENS, D.L.; NELSON, H.A.; RICHARD, J.L. Concentrations of fumonisin B1 in feeds associated with animal health problems. Mycopathologia, v. 114, p. 129-35, 1991. SCHROEDER, J.J.; CRANES, H.M.; XIA, J.; LIOTTA, D.C.; MERRIL, A. H.; Disruption of sphingolipid metabolism and stimulation of DNA synthesis by fumonisin B1. A molecular mechanism for carcinogenesis associated with Fusarium moniliforme. J. Biol. Chem., v. 269, p. 3475-81, 1994. SHEPHARD, G.S. Chromatographic determination of the fumonisin mycotoxins. J. Chrom. A v. 815, p. 31-9, 1998. SHEPHARD, G.S.; THIEL, P.G.; STOCKENSTROM, S.; SYDENHAM, E.W. Worldwide survey of fumonisin contamination of corn and corn-based products. J. Assoc. Of Anal. Chem., v. 79, p. 671-87, 1996. SMITH, G.W.; CONSTABLE, P.D.; EPPLEY, R.M.; TUMBLESON, M.E.; GUMBRECHT, L.A.; HASCHEK-HOCK, W.M. Purified fumonisin B1 decreases cardiovascular function but does not alter pulmonary capillary permeability in swine. Toxicol. Science, v. 56, p. 240-9. 2000. SYDENHAM, E.W.; MARASAS, W.F.O.; SHEPHARD, G.S.; THIEL, F.G.; HIROOKA, E.Y. Fumonisin concentrations in Brazilian feeds associated with field outbreaks of confirmed and suspected animal mycotoxicosis. J. Agric. Food Chem., v. 40, p. 994-7, 1992. WILSON, T.M.; ROSS, P.F.; RICE, L.G.; OSWEILER, G.D.; NELSON, H.A.; OWENS, D.L.; PLATTNER, R.D.; REGGIARDO, C.; NOON, T.H.; PICKRELL, J.W. Fumonisin B1 levels associated with and epizootic of equine leukoencephalomalacia. J. Vet. Diagn. Invest., v. 2, p. 213-6, 1990.

98

_________________ 5 ____
TRICHOTECENES

99

TRICHOTHECENES Summary Trichothecenes (TCT) form part of a group of chemically similar toxic metabolites. They are produced by various fungal species of the Fusarium, Cefalosporium, Myrothecium, Stachybotrys, and Trichoderma genera. More than a hundred mycotoxins form part of this group, which is divided into macrocyclic TCT and non-macrocyclic TCT. The toxic effects of macrocyclic TCT such as baccharinas, roridinas, Trichothecenes production mainly occurs with cool ambient temperatures. satratoxinas, and verrucarinas have yet to be studied in swine. Non-macrocyclic TCT are divided into type A and type B. The main constituents of type A include toxin T-2,

toxin HT-2, and diacetoxyscirpenol (DAS). The main constituents of type B are deoxynivalenol (DON or vomitoxin), fusarenon-X, and nivalenol. TCT occur in various grains and their byproducts, especially those cultivated during winter because the temperatures and high humidity of this season favors the development of the molds which produce these mycotoxins. The mechanism of toxic action of TCT is based on inhibiting the synthesis of protein and interfering with the synthesis of DNA and RNA, affecting cells of high metabolic activity and active multiplication. In addition to feed refusal, TCT induce the formation of ulcerative lesions in the gastrointestinal tract and dermatitis, which is important in clinical diagnosis. Animals present decrease in productivity concurrent with immunosuppression. History In the 1930s, a stachybotryotoxicosis caused the death of tens of thousands of equine in the former USSR. The intoxication was the result of feed contaminated with satratoxins. Ulcerative stomatitis lesions, bleeding, leukopenia, inflammation, and intestinal necrosis are characteristic of this intoxication, and clinical symptoms include blindness and lack of coordination. In the beginning of the XX century and especially between 1941 and 1947, alimentary toxic aleukia (ATA diseases) made a large number of Europeans sick, causing more than 100 thousand deaths in Russia. The illness was related to the ingestion of wheat, rye, and other grains which had been contaminated by TCT produced by cryophilic fungi of the Fusarium genus, especially F. sporotrichoides, graminearum and moniliforme. The four

100 evolutionary stages of the disease occur in the following order: caustic lesions in the upper digestive tract, bloody vomit and diarrhea which last from 3 to 9 days. The second stage occurs for 2 to 8 weeks and is characterized by hematopoietic damage such as leukopenia, agranulopenia and lymphocytosis which decrease resistance to bacterial infections. The third stage is characterized by the formation of petechiae that can reach up to a few square centimeters in size on the skin. These lesions are frequently accompanied by necrotic lesions in the oral cavity which can cause asphyxia and death in approximately 30% of intoxicated animals. The last stage is characterized by dermatitis resulting from direct contact with the toxins. Furthermore, there is a decrease in the productivity of intoxicated animals in every stage of the illness. Etiology TCT is made up of a chemical group of toxic secondary metabolites with the same basic structure (Figures 26 and 27). They are composed of sesquiterpenoids, which are characterized by a 12,13-epoxy-trichothecium-9-ene skeleton type. Table 14 – Main non-macrocyclic trichothecenes. Type Toxins T-2 toxin, HT-2 toxin Diacetoxyscirpenol (DAS), Neosolaniol. Nivalenol, Deoxynivalenol (DON), 4-acetyl-nivalenol (Fusarenon-X), 3-acetyl-deoxynivalenol (3-Ac-DON) Main fungal producers F. sporotrichoides, F. poae, F. oxysporum, F. semitectum e F. equiseti.

A

B

F. graminearum, F. culmorum, F. crookwellense, F. sporotrichoides, F. poae, F. tricinctum e F. acuminatum.
Adapted from Pasteiner (1994) and Pittet (1998).

TCT producing molds belong to species of the Trichoderma, Trichothecium, Myrothecium, Stachybotrys, Cylindrocarpon, Verticimonosporum, Cephalosporium, Phomopsis and especially Fusarium, such as F. graminearum and F. tricinctum genera. The presence of these fungi in food sources does not imply the presence of toxins, but it is an important indicator of potential contamination. Approximately two hundred types of TCT are

101 known; however, only a dozen are known to have toxicological importance. They are divided in two large groups, according to their molecular structure: simple chain, non-macrocyclics (Table 14), and macrocyclics, which have a macrocyclic ring between C4 and C15. TCT macrocyclics are easily distinguishable from non-macrocyclics through thin layer chromatography, because of their intense fluorescence when seen under ultraviolet rays. They are generally more toxic than non-macrocyclics. Frequently, the ingestion of TCT macrocyclics is related to the occurrence of stachybotryotoxicosis and immediate death of animals that have ingested contaminated feed. Non-macrocyclics TCT are divided into four groups: A, B, C, and D. The most important belong to groups A and B. Type B have a group of conjugated carbonyl in position C8, which is absent from type A TCT. The main representative of macrocyclic TCT (type C) are: verrucarins, roridins, satratoxins and baccharins. Crotoxin, a non-macrocyclic diepoxytrichothecene belongs to type D trichothecenes. Figure 26 – Molecular structure of some type A trichothecenes.

CH3

H

O

H O

H OH H

R2

CH3 CH2OCOCH3
R1

R1
R2 OCOCH2CH(CH3)2 OCOCH2CH(CH3)2 OH H

Toxin T-2 Toxin HT-2 Neosolaniol (NEO) 4,15-Diacetoxyscirpenol

OCOCH3 OH OCOCH3 OCOCH3

Type A TCT are soluble in apolar solvents such as chloroform, ethyl acetate, acetone and ether. Type B TCT are soluble in polar solvents, such as water, acetonitrile, and alcohols. Other TCT are normally soluble in solvents used for type A TCT. The resistance of some TCT was tested in grain processing. Experimental corn milling shows that around 67% of T-2

102 toxin was removed. DON proved to be the most stable amongst TCT during food processing given that 50% of the toxin can be lost through baking. It has been found that using sodium carbonate during the elaboration of food increases the loss of toxins. Significant losses of T-2 toxin and DON were observed when extracting oil from contaminated corn. Only traces of T-2 toxin were detected in malt after the fermentation of barley. Deoxynivalenol does not appear to be as unstable during the fermentation process in the production of beer. Out of fifty samples of Argentinean beers analyzed for the presence of deoxynivalenol in 2000, 44% had levels between 4 and 221 ?g/L, with 18% of samples having levels above 20 ?g/L. Generally, TCT are relatively stable at high temperatures. This stability is attributed to the protection of the epoxide ring against nucleophylic attacks. Thus, the melting point of T-2 toxin is 152°C, the melting point of Diacetoxyscirpenol is 164°C, the melting point of Nivalenol a 223°C, and that of Deoxynivalenol is 153°C. Figure 27 – Molecular structure of some type B trichothecenes. The melting point of TCT surpasses 150?C.

CH3

H

O

H O

H R1 H R2

O

OH

H CH3 CH2OH

R1 Nivalenol (NIV) 4-acetyl-nivalenol(Fusarenon-X) Deoxynivalenol (DON) 3-Acetildeoxinivalenol(3-Ac-DON) OH OH OH OCOC

R2 OH OCOCH3 H H

103 TCT of most toxicological and economical importance in the swine industry include deoxynivalenol (vomitoxin; DON) and T-2 toxin, followed by diacetoxyscirpenol (DAS) and nivalenol (NIV).

Occurrence of trichothecenes in feed and food TCT occur globally as a natural contaminant of grains and food sources. Their incidence has been recorded in Asia, Africa, North and South America, and Europe (Table 15). The occurrence of TCT depends on various factors, such as crop strain and its toxigenicity and Contamination by TCT is a major problem in Southern Brazil because of climatic conditions.

climatic conditions, including water activity, preferably above 0.96. This occurs especially when harvest takes place under rainy, cloudy, and cool conditions, with temperatures ranging from 6 to 24°C. Trichothecenes are mostly present in Southern Brazil in grains harvested during winter, when temperatures and humidity levels are adequate for the development of toxin producing fungi. Significant concentrations are frequently detected in oats, barley, corn, wheat, and hay, while low levels of the toxin are usually found in rice, rye, and sorghum. Levels of natural contamination of DON, DAS, T-2 toxin, and nivalenol normally reach up to 10 mg/kg. Levels of up to 15-40 mg/kg are rarely detected. The incidence of DON in food sources is frequent at levels between 0.1- 42 mg/kg, with an average between 2.4 mg/kg and 4 mg/kg. Globally, DON is the most common contaminant of grains, as well as nivalenol (NIV) in certain regions. The concurrent presence of other TCT and other Fusarium toxins such as fumonisins, moniliformin, and zearalenone in the same batch of grain is possible. In 1988 five hundred samples of grains (wheat, barley, rye, corn, and others) from 19 European, Asian, and South American countries were analyzed for NIV and DON by Japanese researchers. Approximately 40 to 50% of the samples were positive for NIV and DON, with an average concentration of Vomitoxin (DON) is the most frequent TCT and is often present with other mycotoxins.

267 and 292 ?g/kg, respectively. Barley had the highest incidence of mycotoxins, being that in 139 of the samples analyzed the average levels of NIV and DON were of 401 and 149,

104 respectively. Of the 222 samples of wheat analyzed, 50 and 39% were positive for NIV and DON, respectively, with an average of 127 ?g/kg and 488 ?g/kg. In samples of wheat and barley from Japan and Korea, levels of NIV were much higher than those of DON, while DON was the principal contaminant in samples from Argentina, Canada, China, Poland, and Germany.

Table 15 – Occurrence of deoxynivalenol (DON) in grains.

Product Wheat Wheat Wheat Corn Wheat Barley Wheat Wheat Wheat Barley

Country Bulgaria Switzerland Switzerland Canada Canada Germany Germany Argentina Argentina Canada

Samples 140 205 154 283 2311 240 445 60 40 117

Incidence 67% 87% 62% 86% 33% 81% 91% 93% 80% 100%

Levels (?g/kg) 50-1,800 20-3,000 10-3,010 20- 4,090 10-10,500 2- 4,764 3- 20,538 100-9,250 300- 4,500 30-15,790
Adapted from Pittet (1998).

In Canada and the United States, the most commonly found TCT are DON, followed by NIV, T-2 toxin, and HT-2 toxin, and rarely DAS. In Argentina, DON is one of the main toxins found in primary food sources, with levels between 200 and 4,500 μg/kg in corn. In other evaluations, Argentinean researchers found DON in 93% of wheat samples analyzed, with average levels of 1,798 μg/kg. Some samples of wheat flour presented contamination between 250 and 9,000 μg/kg with an average of 1,309 μg/kg. To evaluate the occurrence of TCT contamination in wheat, researchers analyzed samples of grain harvested in the main producing areas of Argentina, during a period of 6 years, comprising 1,056 samples. The occurrence of DON was found in 524 (49.6%) samples, with concentrations between 47.5 and 570.9 ?g/kg.

105 Levels of TCT contamination in grains are significantly related with the quantity of plant residue in the grains. Thus, the presence of portions of cob, stalk, stunted grains, and residues can cause and increase in contamination of up to 5 folds. Therefore, there is a tendency to find higher concentrations of toxin in older grains. Approximately 10% of the samples of analyzed grains cultivated in the state of S?o Paulo were found to be positive for TCT. It was also found that concentrations rarely exceed the limit of 1 mg/kg. However, the incidence of TCT in grains cultivated in Southern Brazil is higher, frequently reaching levels higher than 30%. Barley, rye and wheat have the highest incidence of contamination, with average contaminations of 1 mg/kg, reaching levels of up to 15 mg/kg. The incidence of different TCT in feed and feed ingredients from the principal grain producing states of Brazil was analyzed during the last 15 years in LAMIC/UFSM. The incidence of T-2 toxin was analyzed in 16,939 samples. Less than 1% of samples were positive, with an average contamination of 1.2 ?g/kg. The presence of DON was higher, found in 15,809 of the 39,451 samples (40%), with an average contamination of 313 ?g/kg, especially in winter grains such as wheat and barley. Toxicokinetics of trichothecenes The dynamic of absorption, transportation, and distribution by tissues and organs was studied using markers such as 14C. The study is important, especially in order to measure and prevent the risks of human exposure through consumption of contaminated meat products. Absorption Studies testing trichothecenes absorption were done by administering the toxin in three different ways: oral, dermic, and parenteral. The highest concentrations reached through oral exposure are found in the blood stream one hour after administration. The amount of toxin absorbed in the gastrointestinal tract is low, close to 1%, causing local caustic action. Studies done with oral doses in various species show that the maximum radioactive labeled TCT appear in the blood after one hour of being administered. Distribution can be seen in different organs, especially in the liver, kidneys, and in bile.

106 The first reactions to natural exposure are inflammation of the skin, mucosa, and gastrointestinal tract. Absorption of the toxin through the skin is slow and the subcutaneous adipose tissue serves as a reservoir. Among different TCT there is a difference in absorption since the highest concentrations in the tissues and organs can be detected when T-2 toxin is administered. Levels of DON are generally lower because it is largely eliminated through the feces. Distribution The distribution of toxins occurs in different organs and tissues, especially in the liver, kidneys, stomach, and skin. The highest levels of toxin were found in tissues after approximately 3 to 4 hours of initial oral exposure, and after close to 30 minutes when exposure was parenteral. Distribution to adipose and muscular tissue occurred 6 hours after ingestion. In the majority of cases, the toxin was no longer detected 24 hours after administering the contaminated feed. Mechanism of action The adverse effects of trichothecenes are the result of the interaction of the toxin or its metabolites and the target tissue. The largest toxic effects of trichothecenes are a result of interference with protein synthesis, followed by a decrease in nucleic acid TCT are the most potent interferers of protein synthesis.

production. Immediate effects are seen in the function of cell membranes, enzymatic activity, and immune function. The peptidyl transferase enzyme, integral component of ribosomes, responsible for the elongation and termination of the polypeptide chain, is the specific site of action. TCT are also associated with hemorrhages in humans and animals. Prothrombin time significantly increases, thus the primary factor of hemorrhages is a result of a decrease in the VII factor of blood coagulation. They cause lymphoid necrosis in the thymus, spleen, and lymph nodes, and a decrease in the production of immunoglobulin and serum antibodies.

107 Biotransformation Biotransformation consists of a change in molecular structure, generally becoming more polar. Its toxic activity is diminished because there is a reduction in passage through the cellular membrane. Consequently, there is an increase in the excretion of the toxin, especially through the kidney, because of a decrease in reabsoption of nephron levels. Biotransformation occurs as a result of the activation of a series of enzymes located in the endoplasmatic reticulum of various types of cells, especially hepatocytes. Two enzymatic processes take place: the first phase involves oxidation, reduction, and hydrolysis, followed by the second phase in which conjunction reactions and synthesis occur. Like many xenobiotics, TCT are biotransformed biphasically. Three main reactions occur: deacetylation (hydrolysis), hydroxilation (oxidation), and epoxidation (reduction). The hydrolysis of esters is known as the principal metabolic pathways of TCT, containing esterified chains, as occurs in T-2 toxins and diacetoxyscirpenol (DAS), such as the hydrolysis of ester C-4, being the primary sight of attack. Oxidation reactions have only been observed in T-2 toxin. The reduction of bond 12, 23-epoxide by the anaerobic microflora present in TGI is an important detoxification reaction. Elimination The elimination of trichothecenes occurs quickly through feces, but it mainly occurs through urine because of the conjugation of endogenous molecules to the toxin, facilitating elimination. Despite enterohepatic circulation of the toxins, elimination is quick after the removal of contaminated feed. Approximately 75% of the toxin is eliminated within 24 hours, and the toxin is nearly undetected in the bloodstream after 48 hours. The half-life of the toxin in the bloodstream is around 16 hours. Residues TCT are eliminated quickly from the body, hardly detected in animal byproducts, except in adipose tissue. Residues can be found in hen eggs and cow milk. The concentrations of these toxins decrease rapidly when animals resume a diet of uncontaminated feed.

108 Pathogenesis TCT are potent inhibitors of protein synthesis in eukaryotic organisms. Their inhibiting action occurs effectively in different organism, including fungi, plants, and animals. The greatest toxic effect of TCT is a result of the primary inhibition of protein synthesis, by inhibiting the enzymes responsible for the elongation and termination of the polypeptide chain, such as the ribosomal enzyme of the subunit 60S, peptidyl transferase, blocked by the linkage with TCT molecules. Thus, the main mechanism of action is the inhibition of the polypeptide chain by affecting tRNA and subunits of ribosomal cross-linking. Immediate effects are observed in cellular membrane function, enzymatic activity, and immune function. Rapid proliferation cells such as intestinal, mucosa, lymphoid, and erythrocyte cells, are the most affected by inhibition of protein sy

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