Toxicological effects of aflatoxin B1 and fumonisin B1

Toxicological effects of chronic low doses of aflatoxin B1 and fumonisin B1-containing Fusarium moniliforme culture material in weaned piglets

The effects of chronic oral exposure (28 days) to aflatoxin B1 (AFB1) and fumonisin B1 (FB1) were studied in weaned piglets. Six experimental groups, each comprising two neutered males and two females, were fed ad libitum with rations containing: (A) 0 mg of FB1 and 0 mg of AFB1/kg of feed (control); (B) 10 mg of FB1/kg of feed; (C) 30 mg of FB1/kg of feed; (D) 50 mg of AFB1/kg of feed; (E) 10 mg of FB1 plus 50 mg of AFB1/kg of feed; (F) 30 mg of FB1 plus 50 mg of AFB1/kg of feed.

The animals were inspected twice daily and their body weight and feed consumption were recorded weekly and daily, respectively. Samples of feces and urine were collected 24 h after the start of the experiment, to check for fumonisin residues by HPLC analysis. Blood samples were drawn at the start of the experiment and after 28 days for quantification of hematological and biochemical parameters. Necropsies were performed after 28 days; at necropsy, the organs were weighed, inspected macroscopically and processed for histopathological and toxicological analyses.

All piglets from groups C and F presented typical signs of pulmonary edema, with reduced feed consumption and body weight gain as well as pathological alterations. FB1 was detected in feces and urine at 24 h of intoxication and in liver after 28 days of intoxication. Increases were detected regarding the following hematological and biochemical parameters in animals from treatments C and F: erythrocyte number; hematocrit; total bilirubin; total protein; activity of serum alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase.

Cholesterol levels were significantly aumented only in animals from groups C and F, whereas albumin concentrations increased in groups C, F, B and E. The average organ/body weight ratio of piglets (hearth, liver and lung) were significantly greater in groups C and F. The only joint effects of FB1 and AFB1 detected (group F) were a decrease in feed consumption during the last week of intoxication and in feed conversion throughout the 28 days of intoxication.


Chronic intoxication of piglets with AFB1 and FB1 leads to important losses of productivity. # 2003 Elsevier Ltd. All rights reserved.


Keywords: Toxicity; Fumonisin; Aflatoxin; Pig; Porcine pulmonary edema


1. Introduction

Fumonisins are a group of toxic metabolites produced by fungi of the genus Fusarium, especially F. moniliforme and F. proliferatum (Bezuidenhout et al., 1988; Nelson, 1992). These species are natural contaminants of cereals worldwide and are mostly found in corn and its derived products (Shephard et al., 1996). Several naturally occurring fumonisins are known. Fumonisin B1 (FB1) is always the most abundant and toxic metabolite of this group of mycotoxins, representing ca. 70% of the total concentration in naturally contaminated foods and feeds, followed by fumonisins B2 (FB2) and B3. (Murphy et al., 1993; Norred, 1993).

Fumonisins are structurally similar to sphingolipids and their bases, and they inhibit ceramide syntethase, an enzyme in their biosynthetic pathway. This inhibition results in increased levels of sphingoid bases (sphinganine and sphingosine) in serum of exposed animals. Although such alterations are believed to play a major role in fumonisin-induced toxicoses, the mechanism is not totally understood to date (Wang et al., 1991).
Fumonisin B1 is known to be toxic to domestic animals. Equine leukoencephalomalacia (Kellerman et al., 1990) and porcine pulmonary edema (PPE) (Osweiler et al., 1992) are some of the most frequent diseases caused by fumonisins. Body weight and average daily weight gain have been shown to decrease in chicks in parallel with increasing dietary FB1 (Ledoux et al., 1992).

Fumonisins have also been shown to cause hepatocarcinoma and hepatic disease in rats (Gelderblom et al., 2001). Additionally, the occurrence of FB1 in foods has been statistically associated with a high incidence of human esophageal cancer (Rheeder et al., 1992).

Pigs are particularly susceptible to fumonisins, as demonstrated by data from several experimental and naturally acquired outbreaks (Haschek et al., 1992; Colvin and Harrison, 1992; Rotter et al., 1996). FB1 bioavailability can be rather low, roughly 4%; this mycotoxin may affect different organs and tissues, but its greater concentrations are found in liver and kidneys (Prelusky et al., 1994, 1996). The main target organs in pigs are lung, liver, pancreas and heart (Haschek et al., 1992; Osweiler et al., 1992; Smith et al., 2000). Liver hyperplastic nodules and lesions in the distal esophageal mucosa of weaning pigs fed with fumonisins have also been reported (Casteel et al., 1993). Hepatic alterations may be detected by changes in serum biochemistry; the general trend is a progressive increase for enzymes like alkaline phosphatase, sorbitol dehydrogenase, aspartate aminotransferase and gama glutamil transpeptidase. Additionally, the concentrations of cholesterol and bile acids are also significantly elevated in serum (Casteel et al., 1994; Gumprecht et al., 1998).

Aflatoxins B1 (AFB1), B2, G1, and G2 are secondary metabolites produced mostly by certain strains of Aspergillus flavus and A. parasiticus on several agricultural commodities. They may induce a variety of toxic effects in various animals. AFB1 is the most toxic and is hepatoxic, hepatocarcinogenic and mutagenic to humans and several animal species (Newberne and Butler, 1969; Ka¨ renlampi, 1987). Swine are highly susceptible to aflatoxins. Extreme effects can lead to death, but the greatest impact comes from reduced reproductive capability, suppressed immune function, reduced productivity capability and various pathological effects on organs and tissues (Hoerr and D’Andrea, 1983).

Several works have demonstrated since long ago the presence of aflatoxins in cereals and rations. However, after the discovery of fumonisins, reports began to appear on the co-occurrence of these two types of mycotoxins on agricultural products (Chamberlain et al., 1993; Gao and Yoshizawa, 1997; Ueno et al., 1997; Ali et al., 1998). At present little is known about the joint (interactive) toxic effects of aflatoxins and fumo fumonisin on different animal species, although it is accepted that the mechanism of toxicity and the target organs of these toxins are not the same (Wang et al., 1991; Eaton and Gallagher, 1994; Gumprecht et al., 2001).

In view of the current importance and novelty of investigations on the joint biological effects of different mycotoxins that co-occur in feeds, the purpose of this study was to evaluate the time course and dose response of oral intoxication with FB1 and AFB1, and the potential interactive effects of these mycotoxins on weaned piglets.


2. Materials and methods

2.1. Mycotoxins

Mycotoxins administered to piglets were produced in the Laboratory of Mycotoxins of the Institute of Biomedical Sciences (University of São Paulo, Brazil).

Aflatoxin B1 was produced using a toxigenic strain of Aspergillus flavus (IMI-190) obtained from the International Mycology Institute, London, England. Fungal cultures, clean-up of AFB1, quantification, and toxin added to feeds were done according to a previous report (Oliveira et al., 2000).

Fumonisins were produced by a toxigenic strain of Fusarium moniliforme (MRC 286) obtained from South Africa via the ‘‘Programme on Mycotoxins and Experimental Carcinogenesis’’ (PROMEC - Tygerberg, South Africa). Corn cultures of the fungus were prepared as described previously (Alberts et al., 1990) and incubated in a stove for 5 weeks at 25 ºC. Cultures were dried at 50 ºC for 12 h, ground to a powder, assayed for fumonisins, and stored at -18 ºC.


2.2. Preparation of diet

A complete ration was prepared from corn and soybean meal, and a vitamin and mineral premix was added to adjust protein content to 22%. Fusarium culture material containing, respectively, 4.850 and 1.630 mg of FB1 and FB2/kg was added to feeds to achieve the desired concentrations of FB1, never exceeding 0.62% of the basal diet. Two ratios of added Fusarium culture material were used: 10 mg/kg of feed because it provided the concentrations of FB1 and FB2 considered safe for pigs by several workers (Ross et al., 1991), and 30 mg/kg of feed because it provided the concentrations of FB1 and FB2 were frequently detected in foodstuffs in Brazil (Rodriguez-Amaya, 2000). AFB1 was previously mixed with 10 kg of ration to obtain samples of diet at 50 mg aflatoxin/kg of feed; this level of contamination was selected because it corresponds to the maximum concentration of aflatoxin tolerated by the Brazilian Ministry of Agriculture in rations (Brasil, 1988) destined for animal consumption (Rodriguez-Amaya, 2000).

Six dietary treatments were prepared: (A) 0 mg of FB1 and 0 mg of AFB1/kg of feed (control); (B) 10 mg of FB1/kg of feed; (C) 30 mg of FB1/kg of feed; (D) 50 mg of AFB1/kg of feed; (E) 10 mg of FB1 plus 50 mg of AFB1/kg of feed; (F) 30 mg of FB1 plus 50 mg of AFB1/ kg of feed. Multiple feed samples were collected, stored at -20 ºC, and later analyzed. The average frequencies of fumonisins for highest and lowest levels were, respectively, of the order of 26 and 8 mg/kg of FB1 and 8 and 3 mg/kg of FB2. The average level of AFB1 recovery from contaminated diets was 37 mg/kg. Additional analyses of each lot of feed revealed no detectable levels of aflatoxins B2, G1, G2, T-2 toxin, ochratoxin A, zearalenone, vomitoxin, and moniliformin.


2.3. Piglets and treatment protocol

Thirty six clinically normal, 5-week-old (11–12 kg), crossbred (Landrace x Large white x Duroc) weaned piglets were housed individually in concrete-floor indoor pens. They were allowed to acclimatize to their surroundings for 2 weeks, when they were fed with a starter diet and water ad libitum. After the acclimatization period, 24 piglets were selected on the basis of body weight (mean 15 kg), and randomly distributed into one of the 6 treatments groups (two neutered males and two females per group); diet was provided ad libitum.


2.4. Clinical alterations, feed intake and body weight

Piglets were observed twice-daily, and weighed once weekly. Individual feed consumption was recorded daily.


2.5. Fumonisin determination in feces, urine and liver

Twenty four hours after the start of the experiment, samples of urine and feces were collected from all the animals for quantification of fumonisins according to previously described methodologies (Shephard et al., 1992, 1994). The quantification of FB1 in liver on day 29 of the experiment was done according to Thakur and Smith (1996).


2.6. Clinical pathology analyses

At the start of the experiment and after 28 days, blood samples were drawn from the jugular vein for quantification of hematological and biochemical parameters. Samples were analyzed for leukocyte numbers and differential (neutrophils, eosinophils, basophils, lymphocytes and monocytes). The determinations of erythrocyte numbers, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration and total bilirubin were performed as described by Birgel (Birgel, 1982). Serum analyses were done with an automated biochemical analyzer Techinon RA-100 (Bayer, Tarrytown, NY 10591-5097, USA) using commercial kits from Boeringer- Mannheim France SA (Meylan, France), Roche Diagnostics (Mannheim, Deutschland) and Bayer (Bayer, Tarrytown, NY 10591-5097, USA). The biochemical parameters evaluated in serum were total protein, albumin and cholesterol concentrations, and activity of alkaline phosphatase (AP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT).


2.7. Necropsy, gross pathology and histopathology

Necropsy was performed on a female piglet that died on day 23 of the experiment and on the piglets sacrificed on day 29. At necropsy the following organs were weighed: gastro-intestinal tract, full stomach, empty stomach, small intestine, full cecum, empty cecum, full colon, empty colon, heart, liver, gall bladder, spleen, kidneys and lungs. Any macroscopic alterations found in the organs and tissues were recorded; samples of lung, liver, heart, esophagus, stomach, small intestine, large intestine, cecum, colon, pancreas, gall bladder, spleen and kidney were removed, fixed in 10% buffered formalin, (pH 7.8) and processed for microscopical analysis. Thin sections (4 mm) were stained with hematoxilin-eosin (HE), mounted and inspected by optical microscopy.


2.8. Quantification of fumonisins in culture material and feeds

For quantification of fumonisins in Fusarium culture material and piglets diet, all the procedures used for extraction, purification, and detection of FB1 and FB2 by high-performance liquid chromatography (HPLC) were done according to a previous report (Dilkin et al., 2001). Recoveries were performed with corn and cornbased feed samples (n=3) spiked with 0.1, 0.5, 1.0, 5.0 and 10 mg/g. Average recoveries for corn and cornbased feed were, respectively, 92.6 and 88.3% with relative standard deviation (RSD) at 5.04 and 6.22%, for fumonisin B1 and 91.2%, RSD 5.84% and 89.0%, RSD 7.88% for fumonisin B2. Detection limits (S:N=3:1) for corn and corn-based feed were approximately 0.03 mg/g for fumonisin B1 and 0.05 mg/g for fumonisin B2. Confirmation of the identity of the peaks assigned as FB1 and FB2 was made by comparing test chromatograms with standards, with attention to retention, start and end time of peak elution. Samples that presented a peak at the fumonisin retention time were confirmed by addition of standard and reprocessing.


2.9. Statistical analyses

Descriptive statistics (mean and standard deviation) of the dose groups and sex data were first obtained, followed by multifactor analysis of variance (ANOVA) and multiple comparisons with Bonferroni’s test. The level of significance adopted was P<0.05. While comparing data between sexes, P<0.1 was also employed when P<0.05 did not indicate a significant difference.

The data which showed a statistically significant difference between sexes are presented separately. The statistical analyses were done by computer with the software Statgraphics, version 5.1 (Statgraphics Manugistics, Rockville, MD, USA).


3. Results

3.1. Clinical alterations, feed consumption and body weight

The piglets from treatment groups A (control), B (10 mg/kg of FB1), D (50 mg/kg of AFB1), and E (10 mg/kg of FB1 plus 50 mg/kg of AFB1) were clinically normal throughout the experimental period. All the animals from groups C (30 mg/kg of FB1) and F (30 mg/kg of FB1 plus 50 mg/kg of AFB1) presented the same clinical signs characteristic of FB1 intoxication, with similar intensity, and these alterations became more evident between the 20th and the 24th day of the study in all the affected animals.

The piglets were lethargic, had ruffled fur, cyanosis of the sclera, mucosae, and especially ears and tail, increased heart rate, increased respiratory rate with shallow breathing, panting breathing and moist rhonchus; the animals preferentially remained in a posterior decubitus position (sitting dog position). At day 23 of intoxication, one female piglet from treatment group C died after presenting mouth breathing and salivation with foamy saliva in addition to the clinical signs previously mentioned. During the next days (between 25th and 28th), these animals partially refrained from consuming the ration and showed progressive recovery. On day 28, no clinical signs characteristic of PPE were evidenced.

The animals from treatment groups C and F partially refrained from consuming the rations, showing a significant reduction in mean feed consumption during the last week of intoxication as well as in feed conversion throughout the 28 days of intoxication (Fig. 1) and final body weight gain (Fig. 2), when compared to the other groups. An interactive effect of the two toxins was found in group F with respect to both mean feed consumption (week four) and feed conversion (throughout the experiment) (Fig. 1).




Fig. 1. Mean feed consumption at week 4 of intoxication and mean feed conversion after 28 days of intoxication of weaned piglets. (n=4) * The animals were fed ad libitum with rations containing: (A) 0 mg of FB1 and 0 mg of AFB1/kg of feed (control); (B) 10 mg of FB1/kg of feed; (C) 30 mg of FB1/kg of feed; (D) 50 mg of AFB1/kg of feed; (E) 10 mg of FB1 plus 50 mg of AFB1/kg of feed; (F) 30 mg of FB1 plus 50 mg of AFB1/kg of feed. a–c=Different letters represent significant differences by multifactor analysis of variance (ANOVA) in the dose groups data, and multiple comparisons by Bonferroni’s test. The level of significance adopted was P<0.05.


3.2. Residues of fumonisins in feces and urine

All the piglets that ingested fumonisins eliminated the toxin in feces and urine at 24 h after the start of the experiment. The concentrations found in feces were very similar to those of the rations consumed by the animals. The levels of fumonisins detected in urine varied greatly among animals of the same treatment group (Table 1), and FB2 was only found in samples from treated animals, groups C and F, which consumed rations containing the highest mycotoxin concentrations. It was also found (Table 1) that there was a wide variation in mycotoxin concentrations among the samples from the same treatment group, especially in urine levels, as shown by the standard deviation.


3.3. Residues of fumonisins in liver

All the piglets that ingested fumonisins presented detectable levels of FB1 in liver, with wide variations in values within the same treatment group (Table 1). The animals from group F, which were fed with rations containing both fumonisin and aflatoxin, presented the highest concentrations of fumonisins in liver, though such differences with respect to the other groups were not statistically significant.



Fig. 2. Mean weight gain per treatment (n=4) after 28 days of intoxication of weaned piglets. * The animals were fed ad libitum with rations containing: (A) 0 mg of FB1 and 0 mg of AFB1/kg of feed (control); (B) 10 mg of FB1/kg of feed; (C) 30 mg of FB1/kg of feed; (D) 50 mg of AFB1/kg of feed; (E) 10 mg of FB1 plus 50 mg of AFB1/kg of feed; (F) 30 mg of FB1 plus 50 mg of AFB1/kg of feed. a–c=Different letters represent significant differences by multifactor analysis of variance (ANOVA) in the dose groups data, and multiple comparisons by Bonferroni’s test. The level of significance adopted was P<0.05.


Table 1. Mean concentration and standard deviation of fumonisins detected in feces (mg/kg) and urine (mg/l) of weaned piglets at 24 h after the start of intoxication and in liver (mg/kg) after 28 days of intoxication


ND=Non detected; M=male; F=female. The animals were fed ad libitum with rations containing: (A) 0 mg of FB1 plus 0 mg of AFB1/kg (control); (B) 10 mg of FB1/kg; (C) 30 mg of FB1/kg; (D) 50 mg of AFB1/kg; (E) 10 mg of FB1 plus 50 mg of AFB1/kg; (F) 30 mg of FB1 plus 50 mg of AFB1/kg.


3.4. Clinical pathological analyses

The results of the leukogram (number of leukocytes (x106/mm3), neutrophils (%), eosinophils (%), basophils (%), lymphocytes (%) and monocytes (%), mean corpuscular hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin did not reveal any significant difference between the various regimes of intoxication or between those and the control group (data not show).

Mean erythrocyte count, hematocrit, total bilirubin, total protein, AP, AST, ALT, and cholesterol did not differ significantly between animals that ingested ration containing 30 mg of FB1/kg of feed alone (group C) and those exposed to 30 mg of FB1/kg in association with 50 mg of AFB1 (group F), but both groups presented a significant increase when compared to controls and the other treatments. A significant increases in albumin concentration occurred in animals exposed to rations containing 10 or 30 mg of FB1/kg alone or in association with 50 mg of AFB1. No sex-related effect was observed and neither any interactive effect of associated FB1 and AFB1 with respect to the various parameters of clinical pathology evaluated in the present experiment.


3.5. Weight of organs at the end of the experiment

After 28 days of intoxication (Table 2) the mean body/weight ratios for full stomach, empty stomach, small intestine, full cecum, gall bladder spleen, and kidneys did no differ significantly between all the experimental groups or piglet Sexes. On the other hand, mean hearth, liver, and lung body/weight ratios did not differ significantly between sexes but were significantly different in the groups exposed to 30 mg of FB1/kg of ration and 30 mg of FB1 plus 50 mg of AFB1/kg of ration as compared to the other groups. Only gastro-intestinal tract, empty cecum, full colon and empty colon presented significant differences in mean body/weight ratio between sexes. No interactive effect of the association of FB1 and AFB1 was observed with respect to mean weight of organs.


3.6. Gross and histopathological changes

Very evident pathological changes were observed in one piglet (26.3 kg) from treatment C, which died of pulmonary edema at day 23 of intoxication. It presented a clear and foamy fluid in the trachea and bronchi. Golden-yellow fluid (520 ml) was found in the thoracic cavity, which presented rapid coagulation after opening. The lung did not collapse at the time the thorax was opened, nor after its extraction; it weighed 750 g, and had round edges with transudate extravasation over its surface. On the dorsal portion, the edema extended especially to the frontal lobes but was more severe in the lower region, where it practically took over the whole area. The interlobular septa were edematous, 3–4 mm thick, and separated the septa. The walls of the cardiac chambers were flaccid. Histopathology revealed severe inter- and intra-lobular lung edema, widening of interlobular septa with serum infiltration and some mononuclear cells and neutrophils.

The other changes were milder and less frequent: the alveolar lumen contained epithelial cells, erythrocytes and acidofilic fibrillar material; blood capillaries were dilated, with erythrocytes and lymphocytes; some hyaline thrombi could be seen within the alveolar capillaries. The liver was firm and consistent to touch, with dark brown coloration and increased volume and weight (1.1 kg). The hepatic histopathological lesions were mild and few, consisting of hepatomegalocytosis, random hepatocellular necrosis, picnotic or karyorrhetic nuclei, increased number of mitosis and distorted hepatocytes. The presence of enlarged hepatocytes with abundant granular and eosinophilic cytoplasm and eventually increased nucleus distorted the characteristic lobular structure of liver.

The kidneys were found to be congested and weighing 145 g. Upon histopathological examination, no significant renal changes were observed.


Table 2. Mean and standard deviation of organ/body weight ratio (%) of weaned piglets per treatment (n=4) 28 days after intoxication


M=male; F=female. The animals were fed ad libitum with rations containing: (A) 0 mg of FB1 and 0 mg of AFB1/kg of feed (control); (B) 10 mg of FB1/kg of feed; (C) 30 mg of FB1/kg of feed; (D) 50 mg of AFB1/kg of feed; (E) 10 mg of FB1 plus 50 mg of AFB1/kg of feed; (F) 30 mg of FB1 plus 50 mg of AFB1/kg of feed. a–b=Different letters represent significant differences by multifactor analysis of variance (ANOVA) of organ in the dose groups and sex data, and multiple comparisons by Bonferroni’s test. The level of significance adopted was P<0.05*. While comparing data between sexes, P<0.1** was also employed when P<0.05 did not indicate a significant difference.


The animals that also showed clinical signs typical of pulmonary edema (treatments C and F) between days 20 and 24 of intoxication, as well as swine from the other treatment groups, did not present macroscopic pulmonary or cardiac alterations at the end of the experiment (day 29). They only presented a slightly pale liver, which was elastic and resistant to touch. Hepatic histopathological changes were similar to those previously mentioned. The other organs analyzed in the various treatment groups did not show significant gross or histopathological changes.


4. Discussion

Pig breeding has considerable socio-economic importance in the Brazilian combined agriculture and stock raising market. At present, the Brazilian herd is estimated at 37 million animals, with an estimated annual production of 2 million tons of meat that account for 2.13% of the world’s pig breeding. Recent advances in the fields of nutrition, genetics and management have changed the Brazilian pig business into one of the most competitive worldwide (ABIPECS, 2002). Corn is the main component of diets and the annual Brazilian production of this cereal is about 36 million tons, of which approximately 60% are intended for the formulation of poultry and swine feeds. Corn is cultivated essentially in areas with tropical and subtropical climate, where prevailing temperature ranges and humidity levels propitiate the growth of several genera of toxigenic fungi. The estimated losses of this cereal during harvesting and storage and due to damage caused by toxigenic fungi and mycotoxins can reach 25% of overall production (Pedrosa and Dezen, 1991).

Some studies have shown levels of fumonisin and aflatoxin contamination of around 90 and 36%, respectively, in Brazilian corn. The co-occurrence of mycotoxins has already been investigated by several authors and data indicate that the probability of occurrence of aflatoxins and fumonisins at sub-lethal concentrations on the same substrate is quite significant (Rodriguez-Amaya, 2000; Chamberlain et al., 1993). A Brazilian legislation that sets maximum levels of fumonisins in foods and feeds is presently lacking, however the maximum concentration recommended for aflatoxins is 50 mg/kg of feedstuffs (Brasil, 1988). Due to such regulation, that level of contamination as well as lower concentrations were used in the present study. The weaned piglets from a crossbred (Landrace x Large white x Duroc) utilized in this study correspond to the prevailing genetic makeup among swine destined for slaughter in the Brazilian market.

The use of Fusarium moniliforme culture material, frequently employed by other workers (Colvin and Harrison, 1992; Osweiler et al., 1992) for toxicological studies in swine was chosen due to the large amount of FB1 necessary to run the experiment. On the other hand, since the amount of aflatoxin required was smaller, a solution of AFB1 produced from a culture of Aspergillus flavus was employed.

In this work, the first change observed was a drop in feed consumption among piglets of treatments C and F, a feature already reported by Casteel et al. (1994) and Harvey et al. (1995), who recorded diminished consumption of the order of 40 to 60% in swine intoxicated with FB1 at levels of at least 100 mg/kg of feed. After the appearance of clinical signs characteristic of PPE in our animals exposed to the higher doses of fumonisins (treatments C and F), there was a marked reduction in feed consumption and body weight gain.

However, statistically significant differences were found only for feed consumption during the last week of intoxication, with an interactive effect of the two mycotoxins in piglets exposed to 30 mg of FB1 plus 50 mg of AFB1/kg of feed (group F) when comparing group F with group C or either with the other groups, due to the great variation in consumption among intoxicated animals, as also observed by Rotter et al. (1996). The clinical picture that characterized PPE in this work agrees with reports from several other investigators (Haschek et al., 1992; Osweiler et al., 1992; Colvin et al., 1993). However, other toxicological effects such as presence of blood in feces, hemoptysis, and watery diarrhea described by Colvin et al. (1993) and Casteel et al. (1994), were not verified in this study. The lack of significant changes in feed consumption and body weight gain among our animals exposed to lower concentrations of FB1 corroborates the finding of Ross et al. (1991) that doses of FB1 up to 10 mg/kg of feed are safe for pigs, though it is worth mentioning that studies conducted by Colvin and Harrison (1992) and Osweiler et al. (1992) demonstrated a greater resistance to intoxication by fumonisin in young pigs as compared with reproductively active animals.

The recovery of 7 of our 8 animals that presented with clinical signs of PPE after the reduction in dietary intake confirms the findings of Osweiler et al. (1992) and Haschek et al. (1992), who reported 30% recovery of pigs 3–4 four days after withdrawal of contaminated diets. In our study, the percentage of recovered swine was higher even without removal of contaminated feeds, probably due to the low concentrations of mycotoxins employed. The dose of 50 mg of AFB1/kg of feeds was insufficient to reproduce the effects of pig aflatoxicosis and also did not affect dietary intake or body weight of animals that ingested only this mycotoxin. On the other hand, when AFB1 was associated with FB1 (group F), there was an interactive effect with significant differences. That is, piglets from treatment F (FB1 plus AFB1) consumed less feed during the last week of intoxication and presented lower mean body weight gain compared to piglets from treatment C (FB1 only).

Our recovery of high concentrations of fumonisin in feces of intoxicated piglets agrees with the findings of Prelusky et al. (1994), who reported fecal recovery of FB1 in swine at levels greater than 90% of the orally ingested dose, and who ascribed such high fecal concentrations to the enterohepatic circulation. On the other hand, the low rate of absorption of this mycotoxin has some bearing on its toxicokinetic behavior. According to Prelusky et al. (1994) and Prelusky et al. (1996), the recovery of FB1 from urine of orally intoxicated swine can be as much as 2.5% of the ingested dose. Similar concentrations were observed in our study.

While studying the toxicokinetics of FB1, Prelusky et al. (1994) and Prelusky et al. (1996) concluded that the liver is one of the main reservoirs of FB1 and plays an important role in this mycotoxin’s toxicokinetics and toxicodynamics. The FB1 hepatic concentrations presently found corroborate such findings and, in view of this, there should be greater awareness of the risk of fumonisin intoxication due to consumption of liver.

Our dietary concentrations of fumonisins and AFB1 did not significantly affect leukocyte numbers. In the same way, Rotter et al. (1996), when using rations containing up to 10 mg of FB1/kg for a period of 8 weeks, did not find significant hematological changes among intoxicated pigs. Such findings agree with those of Haschek et al. (1992) who employed 166 mg of FB1/kg of feed, Harvey et al. (1995) who used 100 mg of FB1 plus 2.5 mg of AFB1/kg of feed, and Rotter et al. (1996) who used 10 mg of FB1. The trend towards increased numbers of erythrocytes as well as increased hematocrit, albumin, and total serum protein in treatments C and F reflects the effect of dehydration as a function of high concentration of FB1 in animals from treatment groups C and F The increases in activities of AST, ALP and ALT, concentration of bilirubin and cholesterol were statistically significant in groups C and F. These results agree with data from Haschek et al. (1992), Casteel et al. (1993) and Gumprecht et al. (1998). On the other hand, Rotter et al. (1996) were unable to reproduce the same effect in young swine fed with up to 10 mg of FB1/kg.

We also found this to occur in our experiment, as we recorded no increase in serum activities of hepatic enzymes in animals exposed to up to 10 mg FB1/kg, not even after addition of 50 mg of AFB1/kg to feeds. The rise in cholesterol concentrations in our animals exposed to ration with the highest level of FB1 agrees with the findings of Motelin et al. (1994), who described elevated cholesterol levels when pigs were fed rations contaminated with FB1 at concentrations greater than 23 mg/kg.

Changes in the weight of organs of swine intoxicated with FB1 were previously observed by Harvey et al. (1995). These authors reported reduced liver weights and increased lung weights among young pigs intoxicated with 2.5 of AFB1 and 100 mg of FB1/kg of feed for up to 35 days. In the present study, we found no significant differences in mean body/weight ratios of heart, liver, and lungs (Table 2). No interactive effect of these two mycotoxins in group F was detected. Significant differences in mean body/weight ratio of TGI, empty cecon, full colon, and empty colon were the only ones observed between sexes of intoxicated piglets. Rotter et al. (1996) found that male weaned piglets suffered more severe toxic effects that females when exposed to up to 10 mg of FB1/kg of feed. The matter of greater or lesser resistance among male or female swine during their pre or post-reproductively active developmental phase, whether they be neutered or not, deserves more specific and in-depth studies in order to establish their true susceptibility to mycotoxins.

Pathological changes were observed only among swine from treatment C and F that ingested rations containing 30 mg of FB1/kg of feed. Similarly, Rotter et al. (1996) also recorded pathological alterations in swine intoxicated with rations containing 10 mg/kg for a period of 8 weeks. On the other hand, our results disagree with those of Colvin et al. (1993), who found it difficult to induce pulmonary edema in pigs with doses of up to 16 mg of FB1/kg body weight. The gross and microscopic changes observed in the one animal from treatment C that died of pulmonary edema were identical to those described by several authors (Colvin and Harrison, 1992; Colvin et al., 1993; Casteel et al., 1994; Motelin et al., 1994). Nonetheless, the hepatic nodular hyperplasia and esophageal lesions as described by Casteel et al. (1994) were not observed in this work.

Cardiac lesions were only found in the female piglet that died at day 23 of intoxication. According to Smith et al. (2000), pulmonary edema occurs as a consequence of cardiac changes and not because of an increase in lung capillary permeability. The hepatic changes, not always detected, were mild and similar to those reported by Osweiler et al. (1992), Haschek et al. (1992), and Gumprecht et al. (1998). The lack of lesions in the other organs investigated as well as lack of alterations in treatments B, D, and E could be attributed to the low concentrations of mycotoxins used, in agreement with the findings of Casteel et al. (1994).

Based on our results, we may conclude that the dose of 10 mg of FB1/kg of feed is safe for swine, but that 30 mg/kg may cause serious productivity losses and induce PPE among intoxicated animals. Nevertheless, although 10 mg/kg of feed can be considered safe for swine, one should bear in mind that residues of the toxin may be present in products derived from animals exposed to such FB1 levels and eventually cause health hazards to human consumers. The maximum concentration of aflatoxins recommended by the Brazilian Ministry of Agriculture (50 mg/kg) did not have a toxic effect on the animals tested. However, when this concentration was associated with 30 mg of FB1/kg of feed, an interactive effect of the two mycotoxins was observed with respect to feed consumption (week four) and feed conversion.


Acknowledgements

We thank the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq) for financial support to this work. We would also like to thank Cleide Rosana Duarte Prisco, from the Instituto de Ciencias Biome´dicas/USP, for the statistical analysis of the data.


References

ABIPECS, 2002. ‘‘Associacao Brasileira da Industria Produtora e Exportadora da Carne Suına’’. Available at: http://www.abipecs. com.br/estatisticas.asp.

Alberts, J.F., Gelderblom, W.C.A., Thiel, P.G., Marasas, W.F.O., Van Schalkwyk, D.J., Behrend, Y., 1990. Effects of temperature and incubation period on production of fumonisin B1 by Fusarium
moniliforme. Applied. and Environmental Microbiollogy 56, 1729– 1733.

Ali, N., Sardjono, Yamashita, A., Yoshizawa, T., 1998. Natural coocorrence of aflatoxins and Fusarium mycotoxins (fumonisins, deoxinivalenol, nivalenol, and zearalenone) in corn from Indonesia. Food Additives and Contaminants 15, 377–384.

Bezuidenhout, S.C., Gelderblom, W.C.A., Gorst-Allman, C.P., Horak, R.M., Marasas, W.F.O., Spiteller, G., Vleggaar, R., 1988. Struture elucidation of the fumonisins, mycotoxins from Fusarium
moniliforme. Journal of Chemical Society, Chemical Communications 11, 743–745.

Birgel, E.H., 1982. Tecnicas hematologicas de uso corrente em patologia clınica veterinaria. In: Birgel, E.H., Benesi, F.J. (Eds.), Coordenacao de Patologia Clınica Veterinaria. Sociedade Paulista de Medicina Veterinaria, Sao Paulo, pp. 7–23.

BRASIL - Leis e decretos. Ministerio da Agricultura. Portaria MA/ SNAD/SFA n. 07, de 09 de novembro de 1988, Diario Oficial da Uniao de 09 novembro, Secao I, pagina 21.968, Brasılia, 1988.

Casteel, S.W., Turk, J.R., Cowart, R.P., Rottinghaus, G.E., 1993. Chronic toxicity of fumonisin in weanling pigs. Journal of Veterinary Diagnostic Investigation 5, 413–417.

Casteel, S.W., Turk, J.R., Rottinghaus, G.E., 1994. Chronic effects of dietary fumonisin on the heart and pulmonary vasculature of swine. Fundamental and Applied Toxicology 23, 518–524.

Chamberlain, W.J., Bacon, C.W., Norred, W.P., Voss, K.A., 1993. Levels of fumonisin B1 in corn naturally contaminated with aflatoxins. Food and Chemical Toxicology 31, 995–998.

Colvin, B.M., Cooley, A.J., Reaver, R.W., 1993. Fumonisin toxicoses in swine: Clinical and pathologic findings. Journal of Veterinary Diagnostic Investigation 5, 232–241.

Colvin, B.M., Harrison, L.R., 1992. Fumonisin-induced pulmonary edema and hydrotorax in swine. Mycopathologia 117, 79–82.

Dilkin, P., Mallmann, C.A., Almeida, C.C.A., Correa, B., 2001. Robotic automated clean-up for detection of fumonisins B1 and B2 in corn and corn-based feed by high-performance liquid chromatography. Journal of Chromatography A 925, 151–157.

Eaton, D.L., Gallagher, E.P., 1994. Mechanism of aflatoxin carcinogenesis. Annual Review of Pharmacology 34, 135–172.

Gao, H., Yoshizawa, T., 1997. Further study of Fusarium mycotoxins in corn and wheat from a high risk area for human oesophageal cancer in China. Mycotoxins 45, 51–55.

Gelderblom, W.C.A., Lebepe-Mazur, S., Snijman, P.W., Abel, S., Swanevelder, S., Kriek, N.P.J., Marasas, W.O.F., 2001. Toxicological effects in rats chonically fed low dietary levels of fumonisin B1. Toxicology 161, 39–51.

Gumprecht, L.A., Beasley, V.R., Weigel, R.M., Parker, H.M., Tumbleson, M.E., Bacon, C.W., Meredith, F.I., Haschek, W.M., 1998. Development of fumonisin-induced hepatotoxicity and pulmonary edema in orally dosed swine: morfological and biochemical alterations. Toxicologic Pathologic 26, 777–788.

Gumprecht, L.A., Smith, G.W., Constable, P.C., Haschek, W.M., 2001. Species and organ specificity of fumonisin-induced andothelial alterations: Potential role in porcine pulmonary edema. Toxicology 160, 71–79.

Harvey, R.B., Edrington, T.S., Kubena, L.F., Elissalde, M.H., Rottinghaus, G.E., 1995. Influence of aflatoxin and fumonisin B1-containing culture material on growing barrows. American Journal of Veterinary Research 56, 1668–1672.

Haschek, W.M., Motelin, G., Ness, D.K., Harlin, K.S., Hall, W.F., Vesonder, R.F., Peterson, R.E., Beasley, V.R., 1992. Characterization of fumonisin toxicity in orally and intravenously dosed swine. Mycopathologia 117, 83–96.

Hoerr, F.J., D’Andrea, G.H., 1983. Biological effects of aflatoxin in swine. In: Diener, U., Asquith, R., Dickens, J. (Eds.), Aflatoxins an Aspergillus flavus in corn. Auburn University, Auburn, Ala, pp. 51– 55.

Ka¨ renlampi, O.S., 1987. Mechanism of cytotoxicity of aflatoxin B1: role of cytocrome P1450. Biochemical and Biophysical Research Communications 145, 804–860.

Kellerman, T.S., Marasas, W.F.O., Thiel, P.G., Gelderblom, W.C.A., Cawood, M., Coetzer, J.A.W., 1990. Leukoencephalomalacia in two horses induced by oral dosing of fumonisin B1. Onderstepoort
Journal of Veterinary Research 57, 269–275.

Ledoux, D.R., Brown, T.P., Weibking, T.S., Rottinghaus, G.E., 1992. Fumonisin toxicity in broiler chicks. Journal of Veterinary Diagnostic Investigation 4, 330–333.

Motelin, G.K., Haschek, W.M., Ness, D.K., Hall, W.F., Harlin, K.S., Schaeffer, D.J., Beasley, V.R., 1994. Temporal and dose-response features in swine feed corn screenings contaminated with fumonisin mycotoxins. Mycopathologia 126, 27–40.

Murphy, P.A., Rice, L.G., Ross, P.F., 1993. Fumonisin B1, B2 and B3 content of Iowa, Wisconsin, and Illinois corn and corn screenings. Journal of Agricultural and Food Chemistry 41, 263–266.

Nelson, P.E., 1992. Taxonomy and biology of Fusarium moniliforme. Mycopathologia 117, 29–36.

Newberne, P.M., Butler, W.H., 1969. Acute and chronic effects of aflatoxin on the liver of domestic and laboratory animals: a review. Cancer Research 29, 236.

Norred, W.P., 1993. Fumonisins-mycotoxins produced by Fusarium moniliforme. Journal of Toxicology and Environmental Health 38, 309–328.

Oliveira, C.A.F., Kobashigawa, E., Reis, T.A., Mestieri, L., Albuquerque, R., Correa, B., 2000. Aflatoxin B1 residues in eggs of laying hens fed a diet cantaining different levels of the mycotoxin.
Food Additives and Contaminants 17, 459–462.

Osweiler, G.D., Ross, P.F., Wilson, T.M., Nelson, P.E., Witte, S.T., Carson, T.L., Rice, L.G., Nelson, H.A., 1992. Characterization of an epizootic of pulmonary edema in swine associated with fumonisin in corn screenings. Journal of Veterinary Diagnostic Investigation 4, 53–59.

Pedrosa, A.V.B., Dezen, R.B., 1991. O milho: caracterısticas do mercado e perspectivas. Precos Agrıcolas 55, 1–4.

Prelusky, D.B., Trenholm, H.L., Rotter, B.A., Miller, J.D., Savard, M.E., Yeung, J.M., Scott, P.M., 1996. Biological fate of fumonisin B1 in food-producing animals. Advances in Experimental Medicine and Biology 392, 265–278.

Prelusky, D.B., Trenholm, H.L., Savard, M.E., 1994. Pharmacokinetic fate of 14C-labelled fumonisin B1 in swine. Natural Toxins 2, 73–80.

Rheeder, J.P., Marasas, W.F.O., Thiel, P.G., 1992. Fusarim moniliforme and fumonisins in corn in relation to human esophageal cancer in Transkei. Phytopathology 82, 253–257.

Rodriguez-Amaya, D.B., 2000. Occurrence of mycotoxins and mycotoxin- producing fungi in Latin America. In: De Koe, W.J., Samson, R.A., Van Egmound, H.P. (Eds.), Mycotoxins, Phycotoxins in Perspective at the Turn of the Millennium. Wageningen, pp. 309–320.

Ross, P.F., Rice, L.G., Platter, R.D., Osweiler, G.D., Wilson, T.M., Owens, D.L., Nelson, H.A., Richard, J.L., 1991. Concentrations of fumonisin B1 in feeds associated with animal health problems. Mycopathologia 114, 129–135.

Rotter, B.A., Thompson, B.K., Prelusky, D.B., Trenholm, H.L., Stewart, B., Miller, J.D., Savard, M.E., 1996. Response of growing swine to dietary exposure to pure fumonisin B1 during an eightweek period: growth and clinical parameters. Natural Toxins 4, 42– 50.

Shephard, G.S., Thiel, P.G., Stockenstrom, S., Sydenham, E.W., 1996. Wordwide survey of fumonisin contamination of corn and cornbased products. Journal of the Association of Official Analytical
Chemists 79, 671–687.

Shephard, G.S., Thiel, P.G., Sydenham, E.W., 1992. Determination of fumonisin B1 in plasma and urine by high-performance liquid cromatography. Journal of Chromatography 574, 299–304.

Shephard, G.S., Thiel, P.G., Sydenham, E.W., Vleggaar, R., Alberts, J.F., 1994. Determination of the mycotoxin fumonisin B1 and identification of its partially hidrolysed metabolites in the faeces of non - human primate. Food and Chemical Toxicology 32, 23–29.

Smith, G.W., Constable, P.D., Eppley, R.M., Tumbleson, M.E., Gumbrecht, L.A., Haschek, W.M., 2000. Purified fumonisin B1 decreases cardiovascular funtion but does not alter pulmonary capillary permeability in swine. Toxicological Science 56, 240–249.

Thakur, R.A., Smith, J.S., 1996. Determination of fumonisins B1 and B2 and their major hydrolysis products in corn, feed and meat using HPLC. Journal of Agricultural and Food Chemistry 44, 1047–1052.

Ueno, Y., Iijima, K., Wang, S.D., Sugiura, Y., Sekijima, M., Tanaka, T., Chen, C., Yu, S.Z., 1997. Fumonisins as a possible contributory risk factor for primary liver cancer: a 3-year study of corn harvested in Haimen, China, by HPLC and Elisa. Food and Chemical Toxicology 35, 1143–1150.

Wang, E., Norred, W.P., Bacon, C.P., Riley, R.T., Merill Jr, A.H., 1991. Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. Journal of Biological Chemistry 266, 14486–14490.



Authors: P. Dilkin^a, P. Zorzete^a, C.A. Mallmann^b, J.D.F. Gomes^c, C.E. Utiyama, L.L. Oetting^c, B. Correa^a
a Departamento de Microbiologia, Instituto de Ciencias Biomedicas - Universidade de Sao Paulo, 05508-900 Sao Paulo, SP, Brazil
^b Departamento de Medicina Veterinaria Preventiva- Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil
^c Faculdade de Zootecnia e Engenharia de Alimentos - Universidade de Sao Paulo, 13630-970 Pirassununga, SP, Brazil