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Prof. Dr. Alexandre Pires Rosa Universidade Federal de Santa Maria Laboratrio de Avicultura (LAVIC) 97105-900, Santa Maria, RS. Brasil. e-mail: Prof. Dr. Janio M. Santurio Universidade Federal de Santa Maria Laboratrio de Pesquisas Micolgicas (LAPEMI) 97105-900, Santa Maria, RS. Brasil. e-mail: INTRODUCTION Mycotoxins are secondary metabolites produced by fungi that naturally develop in food products, able to cause a wide variety of toxic effects in vertebrate animals, including man (Coulombe, 1991). Exposure to toxins occur mainly by ingestion of contaminated foods, mainly cereals used in feed formulation, as wheat, peanut, sorghum among others (Chu, 1991). Toxigenic fungi can contaminate food in different stages of production and processing, from culture to transportation and storage. In general, mycotoxins have a high chemical stability, which enable them to persist in food even after the usual processing and packaging procedures remove the fungi. The type or chemical structure of mycotoxins depend on the development of specific fungal strains, and their presence in grains and feeds is under the influence of environmental factors such as moisture of the substrate and environmental temperature. Thus, contamination can vary according to environmental conditions, processing methods or production and storage. It also depends on the type of food, as some grains are more susceptible to the growth of certain fungi than others. In a system with high technical level, as in the poultry industry in general, any factor that can negatively affect production will result in extremely high losses to producers. In this context, the importance of natural feed contaminants, such as mycotoxins, should be stressed as they cause considerable damages to poultry production The magnitude of the problem represented by mycotoxin is better demonstrated by an article published in the New Scientist (Mannon & Johnson, 1985), stating that one fourth of the grains produced worldwide are contaminated by mycotoxins. MYCOTOXICOSIS: Mycotoxicosis is the term used to define any disease caused in man and animals by the exposure to mycotoxins. The severity is determined by the mycotoxin involved, the animal species and the effect are dose-dependent and there is an individual variation even within the same species. Mycotoxicosis is characterized by being related to food ingestion, it is neither contagious nor infectious and is always caused by the toxins produced by fungi (Hussein & Brasel, 2001). Diffuse syndromes characterize mycotoxicosis, but lesions predominate in certain organs, as liver, kidneys, epithelial tissue and central nervous system, depending on the type of toxin. However, two or more mycotoxins can be present at the same time, which can potentiate their toxic effects on a susceptible individual. Several mycotoxins have been identified in foods consumed by humans and animals. 1) Aflatoxins The importance of aflatoxins should be stressed not only because they occur very often but also due to the high toxigenic potential that has been shown in production birds. Aflatoxins belong to a group of toxins produced as secondary metabolites by fungi of the Aspergillus genus, especially A. flavus, A. parasiticus and A. nominus (Kurtzman et al., 1987).

The hepatotoxic and hepatocarcinogenic properties of some Aspergillus flavus and A. parasiticus strains were described in the early 60s, followed by the description of the structure of their toxic metabolites, Aflatoxins (AFL). These findings led to a new focus in the studies on mycotoxins, mainly as a result of the problems caused to animal health. The large number of studies and the discovery of aflatoxins were driven by a disease with unknown etiology, that caused high mortality rates in turkeys in England, mainly in poults with levels around 70% and in some cases even 100%. The typical symptoms reported were ingurgitation and renal congestion, with liver hemorrhage or necrosis. Heavy economic losses resulted from the death of hundreds of thousands poults with ages between 4 and 6 weeks. As there was no apparent cause, it was called Turkey X Disease, but symptoms disappeared when the feed was changed. Later on, it was found that the problem was related to the consumption of Brazilian peanut meal contaminated by Aspergillus parasiticus (Santurio, 2000). The growth of A. flavus and production of AFL in its natural substrates are influenced by several factors as mineral components and water activity of the substrate, environmental moisture, temperature and physical damages to the substrate (Viquez et al., 1994). Although high AFL concentrations are found in grains that are stored under hot and humid conditions, it is also possible to detect significant concentrations on the field, before harvest. The concentration capacity is an important characteristic of the AFLs, that is, their levels accumulate along the production chain, as they are highly stable molecules in both biotic and abiotic environments (Quezada et al., 2000). Aflatoxins and their isolated derivatives have more than 20 types of molecules. However, B1, B2, G1 and G2 are still the most studied types (Hussein & Brasel, 2001). The AFLS have a very similar chemical structure, as they are simple chemical compounds with low molecular weight, and a central coumarin nucleus. The B AFLs have a cyclopentane ring in their molecule, while the G series has a lactone ring. The same authors also refer that AFLs are fluorescent substances with unique characteristics. Both aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) have a blue fluorescence, and aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) have a green-yellow fluorescence under UV light. In spite of their structural similarities, AFLs have different degrees of biological activity. Besides being the form most often found in cereals, AFB1 is the most toxicogenic, followed by AFG1, AFB2 and AFG2 (Coulombe et al., 1991). AFB1 is the most toxic both in acute and chronic aflatoxicosis and Aflatoxin M1 (AFM1), a product of detoxification present in milk, resulting from the metabolism of AFB1, is a hepatotoxic substance as potent as AFB1 (Carnaghan et al., 1963). Terao & Ueno (1978) showed that the toxicity of AFG2, AFB2 and AFG1 is equivalent to 10, 20 and 50%, respectively, of the AFB1 toxicity. The European Union established a maximum limit of 10 ppb AFB1 in poultry feed. Countries like Canada, Chile and the United States adopted 20 ppb AFB1 + AFG1 + AFB2 + AFG2 as the maximum limit in the feed for the avian industry (Fonseca, 2003). EFFECT OF AFLATOXICOSIS ON BIRDS One of the most striking characteristics of aflatoxicosis outbreaks is the poor feed absorption, which is manifested by the poorly digested feed particles found in the poultry excreta. It is associated with esteatorrhea or increased lipid excretion (Osborne & Hamilton, 1981), which decreases feed efficiency and, as a consequence, increases production costs. The aflatoxicosis esteatorrhea can be very severe, with a ten-fold increase in the amount of fat in the fecal matter (Schaeffer & Hamilton, 1991). In broilers, there is also a decrease in the specific and total activity of pancreatic lipase, the main fat digestive pancreatic enzyme of fats and by a decrease in bile salts that are necessary both for digestion and absorption of fats. One of the causes for the high toxicity of AFL in birds is its rapid absorption in the gastrointestinal tract, demonstrated by the detection of aflatoxin immediately after the ingestion of mycotoxin (Wyatt, 1991). When absorbed, AFLB1 binds immediately and reversibly to blood albumin

and in a lesser extent to other proteins. The forms of aflatoxin both bound and not bound to serum proteins spread through tissues, mainly the liver. After being deposited in the liver, the absorbed AFLs undergo biotransformation primarily by microsomal enzymes from the oxidative and mixed functions system (Biehl & Buck, 1987). The biotransformation of AFB1 has been studied with great interest, as it maintains a close relationship with its toxic activity mechanisms. There is a consensus among a large number of specialists that AFB1 is in reality a pro-carcinogen that requires metabolic activation in order to manifest its toxic effects (Biehl & Buck, 1987; Hsieh & Atkinson, 1991; Wogan, 1992). The binding of AFB1-epoxide to the DNA modifies its structure thus interfering with the biological activity, originating the basic mechanisms of the mutagenic and carcinogenic effects of AFB1 (Hsieh & Atkinson, 1991). The binding of AFLs to proteins lead to poor liver function with marked alterations in functional properties and protein synthesis (Wyatt, 1991). Primary effects of aflatoxicosis in chickens can be used as an indication for the clinical diagnosis. The first is an alteration in the size of internal organs, increase in liver, spleen and kidneys size, and a decrease in the size of the bursa and thymus. There are also alterations in the color and texture of the organs. The liver, for example, has a characteristic yellow color and is friable, with marked fat infiltration. AFL does not cause mucosa erosions in the gizzard. Occasional lesions can be caused by another mycotoxin. According to Wyatt (1991), besides producing aflatoxins, approximately 36% of Aspergillus flavus strains also produce a mycotoxin called cyclopiazonic acid. A marked pallor of mucosa and legs is also observed in layers that received AFL. This deficiency in pigmentation seems to be the result of a lower absorption, a decrease in transportation and in the tissue levels of carotenoids from the diet (Tyczkowski & Hamilton, 1987; Leeson et al., 1995). In the American poultry industry, aflatoxicosis is known as pale bird syndrome (Figure 1).

Figure 1: Effect of aflatoxin on 21 days of age broilers. Left: Normal bird. Right: Bird with aflatoxicosis: pale bird and liver fat.

Aflatoxins also have an immune suppression effect: aplasia of the thymus and bursa, decrease in the number and activity of T-cells, decrease in the antibody response, suppression of phagocytosis activity and reduction of humoral components as complement (C4), interferon and IgG and IgA immunoglobulins (Pestka & Bondy, 1990; Pier, 1992). All these alterations favor the occurrence of infections, mainly those caused by viral and bacterial agents. A question is raised as to the AFL concentration that is necessary to affect birds performance. The answer is directly related to the animals comfort level, as the higher the stress level, the smaller the

amount of toxin necessary to alter performance (Doerr et al. 1983). There are also factors involved as nutritional imbalance, management failures, extreme temperatures, old litter, quality of chicks being housed. They can all interfere in a decisive manner and low AFL levels in the feed can modify performance. According to Osborne (1982), the minimum concentration of AFL in the diet that can cause losses in the growth of broilers is around 2.5 ppm. Daafalla et al. (1987), on the other hand, found that the administration of 0.5 ppm AFL during four weeks was enough to decrease weight gain in comparison to the control group. The effect of AFL on broilers performance is well demonstrated by Mariani (1998), using 5 ppm in different periods (1 to 7, 1 to 21, 22 to 35, 22 to 42, 35 to 42 and 1 to 42 days of age) and AFLfree diets later on. The most negative and irreversible effect was observed in the initial stage (1 to 21 days). Ghosh et al. (1990) observed that 300 ppb AFB1 in broiler feed causes immune suppression with no apparent clinical effects, but can result in flock morbidity and/or mortality caused by secondary infections. Giambrone et al. (1985a) provided broilers with feeds containing several AFB1 levels during 35 days and found reduction in weight gain and histological liver alterations, but only in the birds that were daily fed levels higher than 500 ppb AFL. In another experiment, however, Giambrone et al. (1985b) did not find signs of aflatoxicosis in chickens fed AFB1 levels up to 800 ppb during 5 weeks. Turkeys submitted to the same treatments had low weight gain and feed conversion rates, as well as an increase in morbidity with different causes and in mortality. The authors concluded that levels up to 66 ppb AFB1 are safe when feeding broilers and turkeys. The results obtained by Kan et al. (1989) in an experiment feeding broilers with 50 and 100 ppb AFB1confirm this statement, as they found no difference between the treatments and the control group. On the other hand, Doerr et al. (1983) performed two experiments with broilers submitted to conditions similar to those of commercial production (0.074m2/bird). In experiment 1, there was a significant reduction in liveweight and carcass weight of birds exposed to feeds containing 75, 225 and 675 ppb AFL in relation to the control group. In experiment 2, however, carried out under the same conditions as experiment 1, there was no significant decrease in the live weight of birds receiving feed contaminated with 300 and 900g/kg aflatoxins. The authors emphasize that when broilers are housed and managed under the same conditions as commercial farms, it becomes difficult to predict a safe feed contamination level, as environmental conditions can cause stress, which potentiates the effects of aflatoxin. Micco et al. (1988) fed young layers during 169 days with 50g/kg AFB1 in the feed and did not observe a reduction in egg production. However, at the end of the experiment, the color of the liver and kidneys were a little lighter than the control group. Oliveira et al. (1999) did not observe either any negative effect on egg production when the layers received less than 500 g/kg AFB1 during an exposure period of 60 days, but also reported significant hepatic lesions in birds receiving levels above 300 ppb. According to Leeson et al. (1995), the main manifestations of aflatoxicosis in layers under experimental conditions include reduction in egg weight and egg production, increased liver fat and alterations in serum enzymes. Hafez et al. (1982) found ovary atresia in experiments with layers receiving feed containing 8 ppm AFB1 during 7 days. Washburn et al. (1985) fed layers with 5 ppm and found a significant decrease in egg weight, which can be explained by the metabolism of aflatoxins that takes place primarily in the liver, responsible for the synthesis and transport of the precursors that are necessary to produce the yolk. This was also observed by Oliveira et al. (2001), feeding layers with AFL concentrations up to 500 ppb with no AFL effect on specific gravity. Oliveira et al. (2002) indicate that the AFB1 levels that were studied (25, 50 or 100 g AFB1/kg) did not alter significantly the specific gravity parameters of quail eggs, in agreement with the

findings of Washburn et al. (1985). These authors reported that there were no alterations in eggs of layers treated with 5000 g/kg aflatoxins (AFB1 + AFB2 + AFG1 + AFG2). Symptoms of the alterations caused by aflatoxins on egg production are not immediate, and are seen after some days or even weeks. The decrease in lay is preceded by the reduction in protein and lipid blood levels. According to Hamilton & Garlich (1971), layers fed 10 ppm AFL have a marked decrease in production, demonstrating that the toxin causes the fatty liver syndrome, affecting the hepatic lipid synthesis, the main component of eggs. Besides reducing egg production, aflatoxicosis is also responsible for the reduction in egg size and the proportional decrease in yolk size, as a result of the interference with protein and lipid synthesis (Huff et al., 1975; Vieira, 1995). The yolk reduction is due to damages to the liver shown in a study by Hamilton & Garlich (1971), where layers fed diets with 10 ppm AFL during three weeks had 48% fat in the liver while layers fed a AFL-free diet had 28%, an evidence of fat accumulation in the liver. As a consequence, there is a decrease in fat deposition in other structures, as in yolk. Several authors refer that calcium deposition on eggshell is not affected by AFL levels in the diet (Wasburn et al., 1985; Hamilton & Garlich, 1971). Huff et al. (1975) did not detect significant alterations in the percentage weight of eggshells from layers fed with AFL in the diets (AFB1 + AFB2 + AFG1 + AFG2). According to Howarth & Wyatt (1976), the transmission of toxins to fertile eggs is the main explanation for losses related to low hatchability. They used diets with 0, 5 and 10 g AFL/g to feed broiler breeders during 4 weeks. They found that fertility was not affected by the different AFL levels, but hatchability decreased significantly in the first week these diets were fed and were 95.1, 68.9 and 48.5% in breeders in the breeders receiving 0, 5 and 10 ppm AFL, respectively. According to Milbradt et al. (2001), the number and weight of hatchable eggs, infertility and hatching were not affected by AFL consumption at 5 ppm concentration during four weeks. Aflatoxins can cause both early and late losses in the performance of broiler breeders. Several economic losses are associated with the ingestion of AFL in the feed, affecting production parameters as decrease in lay (Hamilton & Garlich, 1971), weight loss and decreased feed consumption; and reproductive parameters as low hatchability (Trucksses et al., 1983), decreased egg weight and sperm volume (Leeson et al., 1995). Working with broiler breeders fed diets containing 0, 5 and 10 ppm AFL during 4 weeks, Howart & Wyatt (1976) found that egg production is significantly decreased after 3 and 4 weeks, respectively. In a study with broiler breeders, Rosa et al. (2001) showed that there was a decrease in laying rate after two weeks consuming feed with 5 ppm AFL, but laying went back to normal levels in three weeks when they received a AFL-free diet again. There was no alteration in feed consumption and body weight of infected breeders as a result of the AFL levels used in the study. Trucksses et al. (1983) found AFB1, AFM1 and aflatoxicol in eggs 24 hours after the consumption of a contaminated feed was started. However, it should be stressed that although the laying rate is affected only seven day later, hatchability is already altered 24 hours after the initial consumption of contaminated feed. Jacobson & Wiseman (1974) verified that AFB1 can be transmitted to the eggs of heavy breeders and is deposited in both yolk and albumen. Diets with 100 ppb AFL given to layers during 6 weeks were enough to result in 0.2 ppb toxins in eggs. Aflatoxins can enter the egg at any stage of its development, and it takes 7 to 8 days for an oocyte to mature and another 24 hours to become an egg. As the embryo absorbs the yolk during the third week of incubation, high levels of embryonic deaths are expected during this stage. The levels of aflatoxin in the eggs reach a plateau after 4-5 days and the same time to disappear if the intoxication is interrupted. Qureshi et al. (1998) reported high rates of late embryonic mortality in birds that ingested 5 and 10 ppm AFL, and concluded that the effects were more marked with a longer exposure time to the toxin. This effect is due to the transfer of AFL metabolites or the toxin itself to the egg, causing immune alterations in the embryo.

Occasional problems with embryonic mortality and high rates of early mortality in broilers, commonly seen on the field, are due to the fact that there is a high incidence of viral and bacterial infections. The presence of AFL and its metabolites in hatchable eggs can result in a poor quality progeny. According to Tsukita et al. (2001), the embryonic mortality that occurred in the first and second incubation weeks in eggs produced by the hens fed 5 ppm AFL was not affected by the toxicity. However, mortality became significant during the third incubation week due to the increased mortality in birds that consumed AFL and Deoxynivalenol (DON). Milbradt et al. (2001) reported that the embryonic mortality during the third week is affected by the consumption of diets containing AFL. According to Santurio (2000), the best explanation for hatchability losses of fertile eggs produced by hens that consumed aflatoxins is in the transmission of these mycotoxins to the eggs. Aflatoxin B1 (AFB1) can be transmitted both to the yolk and the albumen. A study was recently carried out in our laboratory (LAVIC UFSM) to determine the effect of toxicity by aflatoxins on the reproductive parameters of broiler breeders and to evaluate the residual effects on the progeny. The study involved 500 females and 60 males Cobb 500: up to 48 weeks of age they were fed AFL-free diets and from 49 to 52 weeks of age the diets contained 0, 250, 500 and 750 ppb of AFL. The 250 to 750 ppb levels did not produce significant effects on laying rate, body weight, specific gravity, shell % and albumen %, fertility and embryonic mortality. It was found that the diet with 250 and 500 ppb of AFL given to broiler breeders had a negative effect on the progeny, as shown in Table 1. A second experiment was performed to analyze the performance of the progeny from the previous experiment breeders. All the chicks received water and feed ad libitum, the diet being the same for all birds and proven to be AFL-free. A marked residual effect of the feed given to the breeders was found in the progeny at 7 days of age (P<0.05). Chicks from breeders fed diets containing AFL had a lower body weight and average daily gain than those from breeders that consumed an AFL-free diet. At 21 days of age, however, the birds performance was no longer affected by the breeders diet. The AFL levels of the study had a significant effect on the broilers mortality (P<0.05), with a linear effect at 1-7 days of age and 1-21 days of age, according to the regression equations: ij=1,418996+18,534323*AFL and ij=2,229214+18,573166*AFL, respectively (Rosa et al., 2005; Fernandes et al., 2005). These data demonstrated a marked relationship between the quality of the diet given to the breeders and the performance of their progeny. These results can also be related to the rapid elimination of AFL and its metabolites. According to Leeson et al. (1995), 28% of the dose is eliminated in the excreta within 24 hours and 71% within 7 days. The aflatoxin effects in broilers are illustrated in the Figures 1 and 2.

Figure 2: Effect of aflatoxin on 33 days of age broilers. Left: Normal bird. Right: Bird with 3ppm AFL.

2) THRICHOTHECENES: The major mycotoxins of this group are: T-2 toxin, Deoxynivalenol (DON) and Diacetoxyscirpenol (DAS), produced by several species of fungi of the Fusarium genus. Studies with chronic toxicity involving T-2 toxin or DAS showed a reduction in feed consumption and weight gain, oral lesions, necrosis of the lymphoid tissue, hematopoietic tissue and oral mucosa, eventual nervous disorders (abnormal wing position, lack of reflexes), abnormal feathering and decreased in egg shell thickness (Burditt et al., 1983, Dziuk et al., 1979, Wyatt et al., 1973, Ademoyero & Hamilton, 1989; Vieira, 1995). Particularly in layers, oral lesions were found in 50% of the flocks when given 2mg/kg T-2, with a parallel decrease in egg production (Diaz et al., 1994). The toxin was shown to be toxic in vitro for poultry macrophage, inhibiting their phagocytosis capacity (Kidd et al. 1995, 1997). T-2 toxin can also form peroxides from lipids, resulting in a decrease in vitamin and concentration in the birds (Hoehler & Marquardt, 1996). Other birds as turkeys and geese are more susceptible to T-2 toxin than broilers (Richard et al. 1978). In geese, egg production starts to decline with 0,1 mg/kg liveweight. The laying and hatchability levels drop by 50% when this toxin is given at a 300mg/kg liveweight level (Vanyi et al., 1994). There are reports in the literature that 3 ppm T-2 toxin in naturally contaminated food was lethal for geese. At around 1 ppm levels in the feed, the T-2 and DAS mycotoxins produce oral lesions in broilers (Wyatt et al. 1973). More significant toxic effects were found at 4 ppm levels with the birds showing low feed consumption, delayed growth, blood alterations and neurotoxicity (Burditt et al. 1983, Wyatt et al. 1973). Oral lesions were also observed in poults with 5 ppm T-2 toxin and weight reduction with 10 ppm. In a direct comparison, it was demonstrated that poults are more sensitive than broilers (Richard et al. 1978). Similar studies with DON have shown that except for a transient decrease in the levels of hemoglobin or a slight effect on egg quality, there is no significant evidence that this toxin affects bird performance (Kubena et al. 1985, 1987a,b, 1989a). It is necessary to stress that trichothecenes are usually produced by Fusarium at temperatures below 15oC. Therefore, special attention should be given to corn originated from Argentina, the US or Southern regions in Brazil above 1,000 meters altitude, as mycotoxins may be present. The T-2 toxin effect is demonstrated in the Figure 3.

Figure 3: Breeder with lesions caused by T-2 toxin.

3) FUMONISINS Fumonisin is produced by the Fusarium verticillioides (moniliforme) fungus. Six types of fumonisins are produced A1, A2, B1, B2, B3 and B4 (Leeson et al., 1995). Fumonisin B1 is the molecular form most often produced and is responsible for triggering the leukoencephalomalacia syndrome in horses and pulmonary edema in swine. Fumonisins are most often detected in corn and corn products. In the North American 1996 harvest, more specifically in the states of Nebraska, Iowa and Illinois, 34.4% of corn samples were found to be contaminated by levels above 0.5 ppm (Carlson & Schneider, 1997). The fumonisins mode of action is related to the blockage of sphingolipid synthesis, an important substance for the cell membrane integrity and ion transport through the cells. Sphingolipids predominate in the central and peripheral nervous system, mainly as the myelin lipid, and are located in the oligodendrocytes and Schwanns cells (Wang et al. 1991). The toxic effects of fumonisin on poultry were first studied by using Fusarium moniliforme cultures as source of this mycotoxin. The culture was given to the birds with the feed. In this way, Weibking et al. (1993a) fed day-old broilers during 3 weeks with feeds containing around 525 ppm fumonisin B1. The birds that received 450 and 525 ppm had a significant decrease in weight gain and feed conversion, increased kidney and liver weight, increased hemoglobin concentration. When compared to the controls, all birds fed fumonisin B1 had high serum levels of sphinganine and sphingosine (precursors of cellular sphingosides). Due to the inhibition of the sphingolipid biosynthesis by the fumonisins, the authors suggest that diets containing more than 75 ppm fumonisin B1 can be toxic for chicks. But these authors did not stress that Fusarium moniliforme produces other mycotoxins besides fumonisins: moniliformin, fusarin, fusaric acid and an unknown number of cytotoxic metabolites. As fumonisin was not purified and the authors used a crude Fusarium moniliforme culture, these results should be seen with reservation. Using turkeys in another test, Weibking et al. (1993b) showed that the deleterious effect of fumonisin B1 was only seen with levels higher than 200 ppm. However, using the methodology similar to that of Weibking et al. (1994), Ledoux et al. (1997) showed that 75 ppm already causes toxicity in poults. Henry & Wyatt (1994) working with purified fumonisin B1, at 0, 20, 40 and 80 ppm, in broilers from 1 to 21 days of age, found no adverse effect on weight gain, feed conversion or water consumption. As to the interactions between Fumonisin B1 and other mycotoxins, there was no synergistic interaction in turkeys (Weibking et al. 1994). However, this synergy was found with moniliformin in poultry (Javed et al., 1993) and with T-2 toxin in turkeys 300 ppm fumonisin plus 5 ppm T-2 (Kubena et al. 1995). This relative innocuity of fumonisins for poultry can be seen as doubtful after the study by Espada et al. (1997), in which they used day-old chicks that were fed 10 mg/kg (ppm) of feed during six days. They found that Fumonisin B1 can contribute to the presence of petechiae, increasing coagulation time and decreasing albumin serum concentration. ENVIRONMENTAL INFLUENCE ON FUNGAL GROWTH a) Moisture Concentration: In practice, the moisture concept is generally sufficient to evaluate the chances for growth and multiplication of fungi in cereals and feeds. But the factor that should always be taken into consideration is the water that is available for the fungus. The parameter to evaluate water availability is called water activity (aw), defined as the relationship between the water vapor tension in a substrate in relation to pure water under the same pressure and temperature. b) Temperature: The temperature for optimal growth is between 25 and 30oC, and the maximum limit is 40 to 45oC. However A. flavus and A. candidus are able to grow even at 55oC. Most fungi do not develop at temperatures lower than 5oC. In a mass of grains or in feed, the temperature is directly related to the environmental temperature, to the heat generated by the metabolism of its microbial load and also by the technical characteristics of the storage site.

The factors that influence fungal growth also interfere with the biosynthesis of mycotoxins and include among others: moisture, temperature, substrate composition, presence of competitive microbiota, as well as the characteristics of the fungus strain. In general, it can be stated that the favorable conditions for fungal growth and multiplication are reached when water activity is higher than 0.75, temperature exceeds 20oC and the substrate moisture is 14% or higher. With a 0.75 water activity at 20oC, which corresponds to 13.5% moisture, fungal spores need 4 to 12 weeks to germinate. However, if the aw of a substrate at 20oC is 0.85, equivalent to 15% moisture, the spores germinate between 5 and 12 days (Santurio, 2003). Krabbe et al., 1994, showed that the increase of the moisture level from 14 to 18% in the corn grain mass stored without aeration during 30 days reduced the crude fat content and, as a consequence, the energy and density (kg/m3) levels by 13.46% and 10.07%, respectively. After 62 days storage, this moisture increase accounted for a 24.46% decrease in the energy level of corn while the decrease in the grain mass density reached -11.72%. MEASURES TO CONTROL MYCOTOXINS: The simple presence or detection of mycotoxins in animal feed does not mean that toxic effects will result. The toxic dose is directly related to the animals sensitivity to the ingested mycotoxin and, in the case of aflatoxins, it is also related to the their comfort level, that is, the lower the stress the more resistant the flock will be to higher levels of aflatoxin. There is no doubt that best method to control contamination by mycotoxins in foods is to prevent fungal growth. The contamination of grains can occur as a result of inadequate storage conditions, but also on the field during the pre-harvest period, and the use of plant genotypes that are more resistant to fungal contamination at storage is very important. Procedures to decrease the harvested grains level of moisture are essential, as well as storage under internationally recommended standards. Fungal growth inhibitors are widely used in stored grains as a preventive measure. The control of fungi activity in feeds and their components has the objective of obtaining raw materials free of mycotoxin production during the storage process (Smith and Hamilton, 1970). However, the growth of fungi in grains and feeds and consequent contamination by mycotoxins can occur in spite of preventive efforts. In this case, the methods for aflatoxin detoxification in food are an alternative. Measures can be taken in order to prevent the consumption of cereals with fungi and mycotoxins by animals, establishing the maximum level of visually altered grains, as grains with fungal growth, broken and with impurities, as well as the maximum level of moisture at receival, 1%. Finally, in order to assure the quality of grains for animal consumption, the nutritionist can monitor the cereals at receival, when the trucks are being unloaded. Several chemicals have been tested and used as fungal inhibitors (Stewart et al., 1977). The main group of these anti-fungal agents are the organic acids, which include substances with simple structures as propionic, acetic, sorbic and benzoic acids and their calcium, sodium and potassium salts (Dixon & Hamilton, 1981). The propionic acid and its derivates, called propionates, are efficient fungal inhibitors and have been used for a long time in poultry feed (Paster, 1979). In 2003, the work by Wilkinson et al. opened new horizons for the control of mycotoxins the use of vaccines. For the first time, these authors were able to induce an immune response to an AFLB1 haptene protein conjugated vaccine. In a not too distant future we will control mycotoxicosis by using preventive vaccines. The use of mineral or organic additives in the diet is another method that helps in the control of mycotoxins in animals, reducing the absorption in the gastrointestinal tract.

CONCLUSIONS: We should always keep in mind that mycotoxins can be a problem even for animals consuming balanced feeds and the solutions to this obstacle to animal production start with a strict selection of raw material, a correct feed storage and monitoring and, when necessary, end with the correct choice of additives that can control the mycotoxins that contaminate the feed in an efficient and safe way.

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