Mycotoxicoses of animals

Mycotoxicoses are intoxications caused by ingestion of foodstuffs contaminated with mycotoxins, i.e. toxic secondary metabolites of microscopic filamentous fungi (moulds). By field observations and by experimental testing, toxins or toxic strains of more than 100 species of fungi have been encountered. However, causal associations have so far only been established for a small number of mycotoxicoses in farm animals, and the more important mycotoxicoses are aflatoxicosis, facial eczema, mycotoxic nephropathy, and estrogenic syndrome. A full assessment of the impact of mycotoxins on the health of farm animals can hardly be made at present, because the amount of surveillance data from mycotoxicoses is very limited, due mainly to inadequate diagnostic criteria. A new set of criteria for the diagnosis of mycotoxicosis is proposed, which has been successfully applied in the causative study of mycotoxic porcine nephropathy.     

Fusarium mycotoxins: a review of global implications for animal health, welfare and productivity

Trichothecenes, zearalenone (ZEN) and fumonisins are the major Fusarium mycotoxins occurring on a worldwide basis in cereal grains, animal feeds and forages. Other important Fusarium mycotoxins include moniliformin and fusaric acid. Spontaneous outbreaks of Fusarium mycotoxicoses have been recorded in Europe, Asia, New Zealand and South America and, in addition, chronic exposure occurs on a regular and more widespread scale. The metabolism and adverse effects of the Fusarium mycotoxins are considered in this review with particular reference to recent data on specific and proposed syndromes and to interactions among co-occurring mycotoxins. Within the trichothecene group, deoxynivalenol (DON) is associated with emesis, feed refusal and depressed feed intake in pigs, while T-2 toxin and diacetoxyscirpenol (DAS) are now clearly linked with oral lesions in poultry. The gut microflora of farm livestock are able to transform DON to a de-epoxy derivative. In contrast, the ovine metabolism of ZEN results in the production of five metabolites and relatively high levels of these forms may be excreted in the urine as glucuronides. There is now undisputed evidence that ZEN and its metabolites possess estrogenic activity in pigs, cattle and sheep, but T-2 toxin has also been implicated in reproductive disorders in farm livestock. Fumonisins are positively linked with pulmonary edema in pigs, leukoencephalomalacia in equines and with deranged sphingolipid metabolism in these animals. Fusarium mycotoxins have also been provisionally implicated in ovine ill-thrift, acute mortality of poultry and in duodenitis/proximal jejunitis of horses. Several Fusarium mycotoxins may co-occur in a particular feed ingredient or in compound feedingstuffs. In general, combinations of Fusarium mycotoxins result in additive effects, but synergistic and/or potentiating interactions have been observed and are of greater concern in livestock health and productivity. Synergistic effects have been reported between DON and fusaric acid; DON and fumonisin B₁ (FB₁); and DAS and the Aspergillus-derived aflatoxins. Limited evidence of potentiation between FB₁ and DON or T-2 toxin has also emerged recently. Additive and synergistic effects between known and unidentified mycotoxins may account for enhanced adverse effects observed on feeding Fusarium-contaminated diets. The potential for transmission of DON into eggs and of ZEN into porcine kidney and liver has been demonstrated. However, lactational carry-over of FB₁ appears not to occur, at least in cows and sows. It is concluded that livestock health, welfare and productivity may be severely compromised by consumption of DON, T-2 toxin, DAS, ZEN and fumonisins and by interactions among these mycotoxins. Safety of some animal products may also be at risk. Furthermore, in view of the limited options available for remediation, it is concluded that exploitation of crops resistant to Fusarium infection offers the most viable strategy for reducing mycotoxin contamination of grain and animal feed.     

Detection and quantitation of T-2 mycotoxin with a simplified protein synthesis inhibition assay.

We describe a simple, rapid, and sensitive bioassay for the detection and quantitation of T-2 mycotoxin by using a protein synthesis assay in cultured cells. Increased sensitivity of the cells to the mycotoxin occurred with time up to ca. 60-min. Time and dose response curves show that an average of 10 to 20 ng of T-2 per ml was sufficient to cause 50% inhibition of protein synthesis in tissue culture cells. A wide range of tissue culture cells with varied type, tissue, and species sources and growth characteristics were tested by this system. All showed approximately the same sensitivity to the mycotoxin. A slight modification of the procedure was used for suspended cultures of mitogen-stimulated lymphocytes, which also showed an equal degree of sensitivity to the mycotoxin. By simply changing the labeled precursor, the inhibition of RNA, DNA, and protein synthesis by T-2 mycotoxin can be compared. Although T-2 mycotoxin had little effect on RNA synthesis, DNA and protein synthesis were equally inhibited. Because of its sensitivity and its capacity to quickly assay a large number of samples, this technique has been a valuable tool in screening samples for the presence of active toxin and has been used to help establish laboratory safety standards for the inactivation of T-2 mycotoxin by chemical agents. It is presently being used in studies of mycotoxin mechanism of action and approaches toward in vivo neutralization of the toxic effects of mycotoxins.     

Zearalenone, deoxynivalenol, and T-2 toxin associated with stalk rot in corn.

The mycotoxins zearalenone (2.8 micrograms/g), deoxynivalenol (1.5 microgram/g), and T-2 toxin (110 ng/g) have been found in the pith of corn stalks standing in the field. Such contaminated stalks may contribute to mycotoxicoses of farm animals.     

Zearalenone, deoxynivalenol, and T-2 toxin associated with stalk rot in corn.

The mycotoxins zearalenone (2.8 micrograms/g), deoxynivalenol (1.5 microgram/g), and T-2 toxin (110 ng/g) have been found in the pith of corn stalks standing in the field. Such contaminated stalks may contribute to mycotoxicoses of farm animals.     

Meta-analytical study of productive and nutritional interactions of mycotoxins in growing pigs

A meta-analysis was carried out in order to study the association of mycotoxins with performance and organ weights in growing pigs. A total of 85 articles published between 1968 and 2010 were used, totaling 1012 treatments and 13 196 animals. The meta-analysis followed three sequential analyses: graphical, correlation and variance–covariance. The presence of mycotoxins in diets was seen to reduce the feed intake by 18% and the weight gain in 21% compared with the control group. Deoxynivalenol and aflatoxins were the mycotoxins with the greatest impact on the feed intake and growth of pigs, reducing by 26% and 16% in the feed intake and by 26% and 22% in the weight gain. The mycotoxin concentration in diets and the animal age at challenge were the variables that more improved the coefficient of determination in equations for estimating the effect of mycotoxins on weight gain. The mycotoxin effect on growth proved to be greater in younger animals. In addition, the residual analysis showed that the greater part of the variation in weight gain was explained by the variation in feed intake (87%). The protein and methionine levels in diets could influence the feed intake and the weight gain in challenged animals. The weight gain in challenged pigs showed a positive correlation with the methionine level in diets (0.68). The mycotoxin effect on growth was greater in males compared with the effect on females. The reduction in weight gain was of 15% in the female group and 19% in the male group. Mycotoxin presence in pig diets has interfered in the relative weight of the liver, the kidneys and the heart. Mycotoxins have an influence on performance and organ weight in pigs. However, the magnitude of the effects varies with the type and concentration of mycotoxin, sex and the animal age, as well as nutritional factors.     

A review of mycotoxin biosynthetic pathways: associated genes and their expressions under the influence of climatic factors

Fungal contamination of agricultural commodities, particularly by mycotoxigenic fungi, represents an enormous concern for global food security in terms of feeding the world's growing population with sufficient and safe food. Not only do they reduce crop yield and quality, but they also produce substantial numbers of mycotoxins, which pose serious adverse health effects in human and animals. As the genome of most mycotoxigenic species have been sequenced, the gene clusters involved in the biosynthesis of agriculturally important mycotoxins including aflatoxins, fumonisins, ochratoxins, zearalenone and trichothecenes, have been largely identified and characterised, with their roles elucidated by researchers. This review provides a comprehensive overview of the current knowledge of genes involved in the biosynthetic pathways of mycotoxins. In addition, the influence of climatic factors including water, temperature and carbon dioxide on differential mycotoxin gene expressions have been highlighted. Overall, the relationship between the relative expression of key regulatory and structural genes under different environmental conditions is significantly correlated with mycotoxins production. This indicates that mycotoxin gene induction can be used as a reliable indicator or marker to monitor mycotoxin production pre-and-post harvest. Furthermore, current strategies to manage mycotoxin risks still require improvement. Thus, an accurate understanding of the molecular mechanisms of mycotoxin biosynthesis in mycotoxigenic species could help to develop an innovative, robust targeted control strategy. This could include the exploitation of novel compounds, which can inhibit biosynthetic genes, to minimise mycotoxin risks.     

The use of trichothecene-contaminated grains in feeds

A review is presented describing the relative efficiencies of the various technologies that have been proposed to permit incorporation of mycotoxin-contaminated grains into animal diets without adversely influencing growth rate or resulting in hazardous residues in edible animal tissues. When the degree of contamination is modest, it may be possible to dilute the contaminated materials with uncontaminated grain to lower the concentration of trichothecenes below the threshold of significant biological activity. A less useful alternative to dilution is the other mechanical approach of milling to remove the most heavily contaminated fractions of the grain. Chemical destruction of triochothecenes is also a possibility. An example is the use of sodium bisulfite treatment to destroy deoxynivalenol in contaminated corn. Such treatments may, however, reduce palatability and nutritional value. When the biochemical mechanism of trichothecene toxicity is known, in vivo therapeutic treatments may be possible. It has been shown, for example, that T-2 toxin-induced changes in brain prostaglandin production can be overcome by treatment with dexamethasone resulting in increased survival. A similar effect was seen using the selective platelet activating factor antagonist BN 52021. Another approach is the use of dietary treatments to either promote in vivo detoxification of mycotoxins or to reduce absorption from the digestive tract with the aid of nonnutritive binding agents.     

Identification of the Toxic Principle in a Sample of Poaefusarin

A sample of poaefusarin (a mycotoxin suspected of being one of the toxins involved in alimentary toxic aleukia in the U.S.S.R.) was received from a Soviet scientist for evaluation and comparison with other mycotoxins. Although poaefusarin is presumed to be a steroid, analyses by thin-layer chromatography, gas-liquid chromatography, and infrared, ultraviolet, and mass spectrometry could not confirm the presence of a steroid structure. However, 2.5% of the sample was made up of the trichothecene T-2 toxin, an amount sufficient to explain the toxicity found in the rat and rabbit skin toxicity tests. In addition, neosolaniol (0.14%), T-2 tetraol (0.6%), and zearalenone (F-2) (0.43%) were present in the sample. Since the toxicity was found to be associated only with T-2 toxin, no attempt was made to determine the nature of the other nontoxic components of the sample.     

Effects of T-2 toxin on ethanol production by Saccharomyces cerevisiae.

A trichothecene mycotoxin, T-2 toxin, inhibits several aspects of cellular physiology in Saccharomyces cerevisiae, including protein synthesis and mitochondrial functions. We have studied growth of, glucose utilization by, and ethanol production by S. cerevisiae and show that they are inhibited by T-2 toxin between 20 and 200 micrograms/ml in a dose-dependent manner. At 200 micrograms/ml, T-2 toxin causes cell death. This apparent inhibition of ethanol production was found to be the result of growth inhibition. On the basis of biomass or glucose consumption, T-2 toxin increased the amount of ethanol present in the culture. This suggests that T-2 inhibits oxidative but not fermentative energy metabolism by inhibiting mitochondrial function and shifting glucose catabolism toward ethanol formation. As T-2 toxin does not directly inhibit ethanol production by S. cerevisiae, this system could be used for ethanol production from trichothecene-contaminated grain products.     

In vitro assessment of adsorbents aiming to prevent deoxynivalenol and zearalenone mycotoxicoses

The high prevalence of the Fusarium mycotoxins, deoxynivalenol (DON) and zearalenone (ZON) in animal feeds in mild climatic zones of Europe and North America results in considerable economic losses, as these toxins affect health and productivity particularly of pigs from all age groups. The use of mycotoxin adsorbents as feed additives is one of the most prominent approaches to reduce the risk for mycotoxicoses in farm animals, and to minimise carry-over of mycotoxins from contaminated feeds into foods of animal origin. Successful aflatoxin adsorption by means of different substances (phyllosilicate minerals, zeolites, activated charcoal, synthetic resins or yeast cell-wall-derived products) has been demonstrated in vivo and in vitro. However, attempts to adsorb DON and ZON have been less encouraging. Here we describe the adsorption capacity of a variety of potential binders, including compounds that have not been evaluated before, such as humic acids. All compounds were tested at realistic inclusion levels for their capacity to bind ZON and DON, using an in vitro method that resembles the different pH conditions in the gastro-intestinal tract of pigs. Mycotoxin adsorption was assessed by chemical methods and distinct bioassays, using specific markers of toxicity as endpoints of toxicity in cytological assays. Whereas none of the tested substances was able to bind DON in an appreciable percentage, some of the selected smectite clays, humic substances and yeast-wall derived products efficiently adsorbed ZON (>70%). Binding efficiency was indirectly confirmed by the reduction of toxicity in the in vitro bioassays. In conclusion, the presented test protocol allows the rapid screening of potential mycotoxin binders. Like other in vitro assays, the presented protocol combining chemical and biological assays cannot completely simulate the conditions of the gastro-intestinal tract, and hence in vivo experiments remain mandatory to assess the efficacy of mycotoxin binders under practical conditions.     

Effect of dietary protein, alfalfa, and zeolite on excretory patterns of 5',5',7',7'-[3H]zearalenone in rats

A series of experiments was conducted to determine how dietary protein, alfalfa, or zeolite influence the excretory patterns of zearalenone (Z), a uterotropic mycotoxin synthesized by Fusarium fungi. Rats were fed diets containing 16.3% casein, 40% casein, 11.2% casein + 25% alfalfa, or 25% casein + 25% alfalfa. Also fed were diets containing 0, 1, 2, or 5% anion exchange zeolite. Tracer doses of [3H]Z were administered either as a constituent of the diet or as a topical application on the skin at the base of the skull. When Z was administered orally, no differences were seen in the fraction of the dose excreted in urine or feces as a result of varying dietary levels of alfalfa and protein. Topical doses resulted in rats fed 25% casein + 25% alfalfa or 40% casein excreting more Z in urine than those fed 25% alfalfa or 16.3% casein. Fecal excretion of Z was greatest for rats fed 25% casein + 25% alfalfa whereas rats fed 40% casein excreted more fecal Z than those fed 16.3% casein. Feeding Z to rats receiving dietary zeolite resulted in a positive correlation between dietary zeolite and fecal excretion of Z but a negative correlation with urinary excretion of Z. Topical administration of Z produced a positive correlation between dietary zeolite and fecal Z excretion but no effect on urinary excretion. It may be concluded that protein and alfalfa treatments alleviate Z toxicosis through increased metabolism whereas zeolite binds Z in the digestive tract to prevent absorption.     

Metabolic effects of trichothecene T-2 toxin

Cereals and other agricultural products contaminated with trichothecene mycotoxins are unfit for consumption. Until recently, the metabolic effects of T-2 toxin (T-2) were thought to reside in its ability to inhibit protein synthesis. It is now clear that trichothecenes have multiple effects, including inhibition of DNA, RNA, and protein synthesis in several cellular systems, inhibition of in vitro protein synthesis, inhibition of mitochondrial functions, effects on cell division, normal cell shape, and hemolysis of erythrocytes. It is argued that these effects are pleiotropic responses of the cell's biosynthetic network to protein synthesis inhibition. However, in studies with erythrocytes, which lack nuclei and protein synthesis, changes in cell shape and lytic response towards T-2 are observed. Susceptibility to lysis is species dependent and correlates with the presence of phosphatidylcholine. Owing to their amphipathic nature, T-2 and other trichothecenes could exert their cytotoxicity by acting on cell membranes. As for cell energetics, T-2 inhibits the mitochondrial electron transport system, with succinic dehydrogenase as one site of action. Although initial investigations of the metabolic effects of T-2 mediated cytotoxicity suggested the inhibition of protein synthesis as the principal site of action, current thought suggests that the effects of trichothecenes are much more diverse.     

Effect of dietary putrescine on whole body growth and polyamine metabolism

Putrescine (1,4-diaminobutane) is the simplest of the mammalian polyamines. These are small, positively charged molecules which are essential for cell growth and are thought to play a role in regulation of anabolic events such as synthesis of DNA, RNA, and protein. Recent reports have indicated the potential for dietary precursor amino acids of putrescine to alter tissue putrescine concentrations. The current study was conducted to determine the physiologic significance of these effects by feeding up to flooding doses of putrescine to determine any influence on whole body growth and polyamine metabolism. A total of 96 chicks were fed purified crystalline amino acid diets containing 0.0, 0.2, 0.4, 0.6, 0.8, or 1.0% purified putrescine (four birds per pen, four pens per diet) for 14 days. The feeding of 0.2% putrescine increased growth rate beyond that of controls while further supplements reduced growth and were toxic when 0.8 and 1.0% putrescine were fed. Hepatic and muscle concentrations of ornithine increased with dietary putrescine while the effect in kidney was much less. Putrescine concentrations in liver, kidney, and muscle rose when 0.4% putrescine or more was fed. This effect was particularly obvious in muscle in which there were also increases in the concentrations of spermidine and spermine. In a subsequent similar experiment, putrescine was fed at 0.0, 0.1, 0.2, 0.3, 0.4, or 0.5% to determine the effect on the activities of the key enzymes regulating polyamine synthesis. The feeding of putrescine at even 0.1% caused a rapid reduction in hepatic ornithine decarboxylase activity while S-adenosylmethionine decarboxylase and arginase activities were not influenced by diet. It was concluded that excess tissue putrescine can be toxic to whole organisms but small, orally administered doses of this metabolite can promote growth.     

Synthesis of a specific protein induced by zearalenone and its derivatives in rat uterus

Zearalenone and its derivatives (alpha-zearalenol and alpha-zearalanol), estrogenic mycotoxins produced by Fusarium species, when added in vivo and in vitro to immature rat uteri, induced the incorporation of labeled amino acids into a specific uterine protein (induced protein). When immature rat uteri were incubated with alpha-zearalenol in vitro, the maximum induction of the induced protein synthesis was obtained with 1 x 10(-6) M and the induction was detected 15 min after the start of the incubation. Moreover, this induction was strongly inhibited by prior addition of inhibitors of RNA synthesis such as alpha-amanitin and actinomycin D. The molecular weight of the induced protein obtained by the in vivo and in vitro treatments with zearalenone and alpha-zearalenol was estimated to be about 52,000 by means of SDS-polyacrylamide gel electrophoresis. These findings clearly indicate that these estrogenic mycotoxins, despite their non-steroidal structures, exhibit an estrogenic activity toward target tissues in a similar manner to that of natural estrogens.     

Development and evaluation of multiplex PCR for detection of T-2 and zearalenone producing Fusarium spp

The study aimed to develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of three major mycotoxin metabolic pathway genes, namely tri8 (T-2 toxin), tri6 (trichothecene) and pks4 (zearalenone), along with competitive internal amplification control. Specific primers for each of the aforementioned genes were optimized and validated using 14 reference strains and 10 pure culture isolates. The optimized mPCR assay detected the three metabolic pathway genes in artificially contaminated maize samples with a sensitivity of 2 × 10³ CFU per g for tri6 and pks4 positive Fusarium strains, whereas 2 × 10⁴ CFU per g for tri8 positive Fusarium strains. Application of the developed mPCR assay to 30 cereal and 20 feed samples revealed 24% (12 of 50) contamination with either one or more mycotoxins. The results of mPCR assay were further evaluated with high performance liquid chromatography (HPLC), and both methods provided unequivocal results. This mPCR assay might be a supplementary tool to conventional mycotoxin analytical techniques like thin-layer chromatography, HPLC, etc. The current mPCR assay is a rapid and reliable tool for simultaneous, sensitive and specific detection of T-2, zearalenone and trichothecene producing Fusarium spp. from naturally contaminated foods and to monitor them during the processing steps of food and feed commodities.     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

Effect of whole and fractionated dietary alfalfa meal on zearalenone toxicosis and metabolism in rats and swine

Experiments were conducted to determine the mechanism by which dietary alfalfa can protect against zearalenone toxicosis. Female weanling rats were fed semipurified diets containing whole alfalfa meal, fractionated alfalfa meal (fiber, solvent extract, and water extract), and purified components of alfalfa (coumestrol, saponin, lignin, coumestrol + lignin, and saponin + lignin) with and without 250 mg zearalenone/kg of diet. All ingredients were provided for 2 weeks at levels corresponding to those found in diets containing 15 and 25% alfalfa. Yorkshire gilts were fed 15 and 25% alfalfa meal with and without 10 mg zearalenone/kg of diet for 4 weeks. The feeding of zearalenone to rats reduced growth and food consumption but this was overcome by 25% alfalfa. Zearalenone also increased the activity of hepatic 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), the enzyme believed to metabolize zearalenone to alpha- and beta-zearalenols. Dietary alfalfa did not overcome this effect. Alfalfa fiber was the only fraction to partially overcome the growth-depressing effects of zearalenone while the other fractions had no beneficial effects and 3 alpha-HSD was not affected by diet. None of the purified components affected growth parameters or 3 alpha-HSD. The enzyme was also not affected by zearalenone or alfalfa in swine diets. Coumestrol, alpha-zearalenone, and beta-zearalenone were shown to be competitive inhibitors of 3 alpha-HSD in rat liver. It was concluded that the fiber fraction of alfalfa protects against zearalenone toxicity, and that this effect is not dependent on coumestrol or saponin and is not likely mediated through 3 alpha-HSD.     

Solvent comparison in the isolation, solubilization, and toxicity of Stachybotrys chartarum spore trichothecene mycotoxins in an established in vitro luminescence protein translation inhibition assay

It is well known that non-viable mold contaminants such as macrocyclic trichothecene mycotoxins of Stachybotrys chartarum are highly toxinigenic to humans. However, the method of recovering native mycotoxin has been without consensus. Inconsistencies occur in the methods of isolation, suspension, preparation, and quantitation of the mycotoxin from the spores. The purpose of this study was to provide quantitatively comparative data on three concurrent preparations of 10(6)S. chartarum spores. The experiments were designed to specifically evaluate a novel method of mycotoxin extraction, solubilization, and the subsequent inhibitory effect in an established in vitro luminescence protein translation assay from 30 day-old spores. The mycotoxin-containing spores swabbed from wallboard cultures were milled with and without glass beads in 100% methanol, 95% ethanol, or water. Milled spore lysates were cleared of cell debris by filter centrifugation followed by a second centrifugation through a 5000 MWCO filter to remove interfering proteins and RNases. Cleared lysate was concentrated by centrivap and suspended in either alcohol or water as described. The suspensions were used immediately in the in vitro luminescence protein translation assay with the trichothecene, T-2 toxin, as a control. Although, mycotoxin is reported to be alcohol soluble, the level of translation inhibition was not reliably satisfactory for either the methanol or ethanol preparations. In fact, the methanol and ethanol control reactions were not significantly different than the alcohol prepared spore samples. In addition, we observed that increasing amounts of either alcohol inhibited the reaction in a dose dependent manner. This suggests that although alcohol isolation of mycotoxin is desirable in terms of time and labor, the presence of alcohol in the luminescence protein translation reaction was not acceptable. Conversely, water extraction of mycotoxin demonstrated a dose dependent response, and there was significant difference between the water controls and the water extracted mycotoxin reactions. In our hands, water was the best extraction agent for mycotoxin when using this specific luminescence protein translation assay kit.