Effect of the appetite stimulant cyproheptadine on deoxynivalenol-induced reductions in feed consumption and weight gain in the mouse.

Deoxynivalenol (DON, vomitoxin), a Fusarium mycotoxin, is suspected of inducing its anorectic/feed refusal activity through a serotoninergic (5HT) mechanism, possible via 5HT2-receptors. In this study the efficiency of cyproheptadine (CYP), a serotonin antagonist and known appetite stimulant, to attenuate the adverse effect of DON was investigated in mice. CYP was administered in the feed for two days before animals began receiving the DON, which was also added to the feed. Both agents were administered concurrently thereafter for a 12-day period. Dosing levels included various combinations of the two compounds, ranging from 0-16 ppm DON and 0-20 ppm CYP.

The role of tryptophan in DON-induced feed rejection.

Deoxynivalenol (DON, vomitoxin) commonly produced by Fusarium fungi can alter feeding behaviour of pigs and other farm animals. The effects of dietary tryptophan (TRP, precursor of brain amine serotonin) in combination with DON were examined in mice to determine if TRP can modulate DON toxicity. Results indicated that brain TRP can be influenced by dietary TRP, but no evidence of TRP potentiating DON toxicity was observed. Higher TRP levels likely induced amino acid imbalance leading to weight gain suppression.

Toxicology of deoxynivalenol (vomitoxin).

Trichothecene mycotoxins are a group of structurally similar fungal metabolites that are capable of producing a wide range of toxic effects. Deoxynivalenol (DON, vomitoxin), a trichothecene, is prevalent worldwide in crops used for food and feed production, including in Canada and the United States. Although DON is one of the least acutely toxic trichothecenes, it should be treated as an important food safety issue because it is a very common contaminant of grain. This review focuses on the ability of DON to induce toxicologic and immunotoxic effects in a variety of cell systems and animal species. At the cellular level, the main toxic effect is inhibition of protein synthesis via binding to the ribosome. In animals, moderate to low ingestion of toxin can cause a number of as yet poorly defined effects associated with reduced performance and immune function. The main overt effect at low dietary concentrations appears to be a reduction in food consumption (anorexia), while higher doses induce vomiting (emesis). DON is known to alter brain neurochemicals. The serotoninergic system appears to play a role in mediation of the feeding behavior and emetic response. Animals fed low to moderate doses are able to recover from initial weight losses, while higher doses induce more long-term changes in feeding behavior. At low dosages of DON, hematological, clinical, and immunological changes are also transitory and decrease as compensatory/adaptation mechanisms are established. Swine are more sensitive to DON than mice, poultry, and ruminants, in part because of differences in metabolism of DON, with males being more sensitive than females. The capacity of DON to alter normal immune function has been of particular interest. There is extensive evidence that DON can be immunosuppressive or immunostimulatory, depending upon the dose and duration of exposure. While immunosuppression can be explained by the inhibition of translation, immunostimulation can be related to interference with normal regulatory mechanisms. In vivo, DON suppresses normal immune response to pathogens and simultaneously induces autoimmune-like effects which are similar to human immunoglobulin A (IgA) nephropathy. Other effects include superinduction of cytokine production by T helper cells (in vitro) and activation of macrophages and T cells to produce a proinflammatory cytokine wave that is analogous to that found in lipopolysaccharide-induced shock (in vivo). To what extent the elevation of cytokines contributes to metabolic effects such as decreased feed intake remains to be established. Although these effects have been largely characterized in the mouse, several investigations with DON suggest that immunotoxic effects are also likely in domestic animals. Further toxicology studies and an assessment of the potential of DON to be an etiologic agent in human disease are warranted.

Response of growing swine to dietary exposure to pure fumonisin B1 during an eight-week period: growth and clinical parameters.

Consumption of corn or corn-based products contaminated with Fusarium moniliforme/fumonisins has been associated with a variety of animal and human diseases and is a major food/feed safety issue. This study focused on the clinical toxicity and performance parameters in growing swing exposed to low to moderate levels of pure fumonisin B1 (FB.) for 8 weeks. Male (castrated) and female pigs were fed diets containing 0,0.1,1.0, and 10 mg FB1/kg diet (ppm). Weight gains and feed consumption were measured weekly. Blood samples were collected throughout the study, and various clinical and hematological parameters were measured. Because fumonisins are potent inhibitors of sphingolipid biosynthesis, sphinganine and sphingosine concentrations were determined in the liver, lung, and kidney. Organ weights and carcass quality were measured at the end of the trial. In general, male pigs were more adversely affected by FB1 in the diet than females. The average daily gain for males decreased by 8% for pigs fed 1.0 ppm and by 11% at 10.0 ppm, when compared to the control (0 ppm). Males fed 0.1 ppm showed an erratic growth pattern during the first 5 weeks of the experiment. Feed consumption for the same animals was somewhat higher than that of the controls during each of the first 4 weeks but thereafter was 6-7% lower each week as compared to controls. Female pigs fed FB1-diets showed a general enhancement of feed consumption until week 4. Among clinical chemistry parameters, cholesterol increased in males for the 1.0 and 10.0 ppm diets as compared to controls after 2 weeks, while the levels in both sexes were elevated for the 1.0 ppm diet only by the end of the experiment. Serum liver enzyme concentrations were altered during week 2 only. Changes were observed in the weight of the pancreas and adrenals for male pigs fed FB1 diets as compared to controls. The free sphinganine to free sphingosine ratio (biomarker of exposure in FB1-consuming animals) increased in all three organs for the 10 ppm diet, regardless of sex. The study indicated that FB1 can cause different effects at each dose level, at concentrations as low as 0.1 ppm (showing erratic growth) followed by a reduced growth and biochemical abnormalities in blood (1.0 ppm) and sphingolipid alterations in tissues (10.0 ppm). Some of these effects occurred below the exposure level that caused alteration in sphingolipid metabolism.

Biological fate of fumonisin B1 in food-producing animals.

The presence of mycotoxins in grains and feedstuffs causes not only animal health problems, but also a valid concern about the transmission of potentially toxic residues into animal-derived products intended for human consumption. In a series of studies at Agriculture and Agri-Food Canada, we investigated the biological fate of fumonisin B1 (FB1) in several food-producing animals (grower pigs, laying hens, dairy cattle), as well as monitored various parameters for evidence of toxicity in these species. In several experiments involving either single-dose protocols (iv, po) or longer-term feeding trials, the pharmacokinetic profiles of FB1 (purity > 95%) in these species were determined, including tissue accumulation and transmission of residues. Toxicological (and economical) implications such as performance (feed consumption, growth), productivity, and carcass quality were also measured when appropriate.

Mycotoxin fumonisin B1 stimulates nitric oxide production in a murine macrophage cell line.

Fumonisin B1 (FB1), a mycotoxin, is a common fungal contaminant of corn and corn products. This sphingolipid-like compound causes a variety of animal diseases and is a suspected human carcinogen. Cellular targets of FB1 include hepatocytes and renal and immune cells. The effects of FB1 on nitric oxide (NO) production induced by lipopolysaccharide (LPS) were investigated in the present study by using a murine macrophage cell line as a model system. Stimulation of NO production was observed for increasing concentrations of FB1 (1, 10, and 100 microM) and either 0.005 or 0.01 microgram/ml LPS. Although with an increasing dose of FB1 the total protein content decreased for the stimulated and the unstimulated cells, the NO production remained elevated for the stimulated cells. It can be hypothesized that the potentiation of the LPS-dependent NO production by FB1 treatment could be due to direct interaction between FB1 and NO synthases and LPS receptors or to a disrupted sphingolipid metabolism.

Influence of low-level exposure to Fusarium mycotoxins on selected immunological and hematological parameters in young swine.

The effects of low dietary concentrations of Fusarium mycotoxins (deoxynivalenol (DON), 15-acetyl-DON, and zearalenone) on growth, immunological, and hematological parameters were determined in young pigs during a 28-day feeding experiment. Clean and naturally contaminated corn were incorporated into basal diets formulated to contain 0.00, 0.75, 1.50, and 3.00 mg DON/kg diet. A pair-fed control animal was used for comparison with each animal receiving the highest level of contamination (diet 4). Skin temperature, measured during the first week of the experiment, decreased linearly as the dietary mycotoxin concentration increased. Several other linear effects were observed: depressed feed intake throughout the experiment, reduction in thyroid size (absolute/relative), and changes in the appearance of the esophageal region of the stomach (thicker and higher degree of folding with increasing toxin concentration). Serum T4 (thyroxine) levels increased quadratically after 7 and 28 days of exposure compared to control animals. This change coincided with an increase in albumin levels, a decrease in alpha-globulin levels, and an overall increase in albumin/globulin ratio as the level of contamination increased. After immunization with sheep red blood cells (SRBC), animals fed contaminated diets showed a delayed response in peak titers. At the end of the experiment an increase in the segmented neutrophil count was observed. The following observations were made for animals consuming diet 4 as compared to the pair-fed controls: lower skin temperature, better feed efficiency, more corrugated stomachs, reduced alpha-globulin levels, and lower antibody titers to SRBC.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of low-level dietary deoxynivalenol on haematological and clinical parameters of the pig.

The subacute toxic effects of dietary deoxynivalenol (DON) were examined in grower pigs during a 32 day feeding period. DON was incorporated into the feed at 0, 1, and 3 mg/kg, added as either the purified toxin (P) or as naturally contaminated corn (N). Growth performance and blood biochemical and haematological parameters were monitored throughout the study. At the higher toxin levels (diets 3P, 3N) significantly lower feed consumption and body weight gains were evident soon after the start of feeding, but while weight gains of pigs fed the pure DON diet (3P) recovered after several days, values for pigs fed the naturally contaminated diet (3N) remained depressed over the course of the study. It is possible that these observations reflected the presence of other unidentified toxic compounds in the naturally contaminated grain. Generally, blood chemistry parameters of pigs fed the contaminated diets were not different from controls, with the exception of alpha-globulin and possibly cortisol in animals receiving diets 3N or 3P. Data suggested that the effect of DON on the alpha-globulin fraction may have been independent of the feed refusal syndrome associated with this toxin. Alterations in several haematological measurements were noted to occur sporadically with the 3 ppm diets, including higher RBC count, haematocrit and platelet level, however these effects could not be separated from the influence of decreased feed intake and were of limited value in diagnosing the effects of low level dietary DON on swine.

Effect of enzyme supplementation on the nutritional value of raw, autoclaved, and dehulled lupins (Lupinus albus) in chicken diets.

The effects of adding crude enzyme preparations to diets containing raw, autoclaved, and dehulled lupin seeds on the performance of broiler and Leghorn chicks (7 to 21 days) were evaluated in five experiments. In the first experiment, enzyme addition (combination of Energex-carbohydrase, Bio-Feed Pro-protease, and Novozyme-alpha-galactosidase) to a diet containing 70% raw lupins improved the weight gain and feed to gain ratio of broiler chicks by 18 and 10%, respectively. The relative intestinal length and gizzard weight also were reduced by enzyme treatment. In the second experiment, the optimum concentration of enzymes was determined in diets containing 50% raw lupins. Bio-Feed Pro at 3% increased weight gains by 24% and the feed to gain ratio by 11%, whereas a combination of three enzymes at .10% of each yielded respective improvements of 18 and 9%. In Experiment 3, the AME and protein digestibility of dehulled lupin seeds were increased by 18 and 7% compared with those for raw seeds. Autoclaving (20 min) significantly (P < .05) improved chick performance and AME and protein digestibility of raw seeds. In the fourth experiment, autoclaving (15 min), dehulling, and a combination of both improved weight gains by 11, 15, and 8% and feed to gain ratios by 4, 11, and 6%, respectively. Enzyme addition improved the performance of birds fed raw, but not autoclaved lupin diets. In the fifth experiment, dehulling and enzyme treatment but not autoclaving (20 min) improved the performance of birds fed diets containing 50% lupins. The relative weight of gizzard was reduced by both treatments but that of the pancreas was affected only by enzyme treatment. Overall, enzyme supplementation of raw lupin diets considerably improved chick performance. Dehulling of lupins also improved chick performance with results for autoclaved lupins being inconsistent.

Rapid colorimetric bioassay for screening of Fusarium mycotoxins.

The cytotoxicity of Fusarium mycotoxins was evaluated using a trichothecene sensitive cell line (BHK-21, baby hamster kidney cells) in combination with the MTT-cleavage test as an end-point measurement. Cells tended to be more sensitive to the type A trichothecenes with midpoint cytotoxicity values ranging from 1.6 ng/ml for T-2 toxin to 60 ng/ml for scirpentriol. The cytotoxicity value for deoxynivalenol (type B) was 112 ng/ml. The inherent disadvantage of the MTT-assay (formation of insoluble formazan) was overcome by using the analog MTS and measuring the water-soluble formazan directly in the culture media. The MTS-midpoint cytotoxicity values for T-2 toxin and deoxynivalenol (2.1 and 141 ng/ml, respectively), although slightly higher, showed a good correspondence to the MTT-test. Both the MTT- and MTS-cleavage tests are useful for evaluating the cytotoxicity of Fusarium mycotoxins. The replacement of MTT by MTS substantially reduced the number of sample processing steps and the length of time required to complete the cytotoxicity assay.

Investigations in the use of mice exposed to mycotoxins as a model for growing pigs.

A series of experiments was conducted to determine the feasibility of using mice to screen for possible dietary mycotoxin interactions before testing them with swine. Selected mycotoxins, deoxynivalenol (DON) and T-2 toxin, were fed to young mice, alone and in combination. The severity of effects on body weights caused by DON (0-20 mg DON/kg diet) was more pronounced in a dose-related manner when the animals were exposed to contaminated diets starting at 21 d of age than at 28 d (Experiment 1) as reflected in the analysis of variance. The relative variance among diets after 7 d was twice as great for the younger than for the older mice. In both age groups, the weight gain response was linear, similar to that seen in growing swine. In Experiment 2, a significant (p < .05) diet type x DON interaction for food consumption evident after 7 d, indicated that the effect of DON depended on the type of diet (freeze-dried vs. regular mash). There was no difference in food efficiency between diet type, but a strong dose-dependent effect due to DON was observed. When DON and T-2 toxin were fed together to young mice, a significant (p < .001) linear decrease in weight gain and food consumption was observed after 7 d on the contaminated diet as the toxin concentration increased.

The effect of enzyme supplementation on the apparent metabolizable energy and nutrient digestibilities of wheat, barley, oats, and rye for the young broiler chick.

The influence of enzyme supplementation on the bioavailable energy (AME(n)) and apparent digestibilities of lipid (ALD) and protein (APD) in young broiler chicks was examined for diets containing either wheat, hulled or hulless barley, naked oats, or spring rye. Dietary AME(n), APD, and ALD values were depressed (P less than or equal to .01) for all test grains (except hulled Bedford barley) as the inclusion rate of the grain replacing wheat increased. The antinutritives, beta-glucans (barley and oats) and pentosans (rye), had the most pronounced effect on ALD. The decreases in ALD were 43, 77, and 67% for chicks fed diets containing 70% Scout barley (hulless), Terra oats, and Gazelle rye, respectively, compared with those fed the control wheat diet. Enzyme supplementation increased (P less than or equal to .01) AME(n), APD, and ALD for all test cereals. The corresponding increases in the AME(n), of the enzyme-supplemented diets containing 70% HY320 wheat, Bedford barley, Scout barley, Terra oats, and Gazelle rye diets were 4, 7, 42, 33, and 14%, respectively, compared with their unsupplemented counterparts. Enzyme treatment also improved (P less than or equal to .01) weight gains and feed conversion efficiencies of chicks fed diets containing each of the cereals. Overall, the results demonstrate that the nutritive value of cereal grains such as wheat, barley, oats, and rye can be improved by the addition of crude fungal extracts to the diet of young chicks.

The effects of enzyme supplementation on the nutritive value of rye grain (Secale cereale) for the young broiler chick.

Two dose-response experiments utilizing differing enzyme concentrations were carried out to determine the level of a crude enzyme preparation (Trichoderma viride cellulase) required to obtain maximum growth performance in young broiler chicks fed a 60% rye diet. Enzyme concentrations utilized in these studies ranged from .05 to 12.8 g enzyme/kg diet. Enzyme supplementation at the 3.2 g/kg diet level yielded increases in feed consumption and weight gain of 71 and 193%, respectively; and the feed to gain ratio was improved by 43% compared with that for the unsupplemented rye control diet. As no further growth response was obtained by increasing the enzyme concentration, the enzyme level utilized in a subsequent study was 3.2 g/kg diet. A balance study using 2-wk-old broiler chicks was carried out to determine the effect of enzyme supplementation on the bioavailable energy (AMEn) and apparent protein digestibility (APD) of a 60% wheat diet replaced by rye in 10% increments. In the 60% rye diets, the enzyme addition increased the dietary AMEn and APD by 23 and 12%, respectively, compared with the unsupplemented diet. The AMEn and APD of the 60% wheat control diet were increased 9.7 and 4.6%, respectively, by enzyme addition. The data suggest that the AMEn of rye grain is increased by enzyme supplementation; however, alternate methodology will have to be employed to quantitatively determine the extent of these improvements.

Evaluation of potential interactions involving trichothecene mycotoxins using the chick embryotoxicity bioassay.

The Chick Embryotoxicity Screening Test (CHEST) was used to examine possible interactions between different trichothecenes. Combinations of deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON) and HT-2 toxin were tested at various concentrations and the effects were determined based on percent mortality. The combined toxicity of any two trichothecenes was found to be additive. Discrepancies between observed and expected mortality values seldom differed by more than 10%, well within sampling error for the binomial distribution. Several limitations of the CHEST assay are discussed.

Estimation of nitrogen-corrected true metabolizable energy of two different barley samples with and without enzyme supplementation over different excreta collection times.

The TMEn values of two barley samples and a corn sample with and without enzyme addition were determined in an experiment conducted over two consecutive weekly periods. Adult roosters were not influenced by the high beta-glucan content of Scout barley, which had a higher energy value (3.70 kcal/g DM) than the Bedford barley (3.44 kcal/g DM). Overall the energy value of the grain increased 3% due to enzyme supplementation. The 48-h excreta collection time was considered to be adequate for TMEn determination.

Estimation of apparent protein digestibility using uric acid-corrected nitrogen values in poultry excreta.

Apparent protein digestibility values were calculated using three methods based on nitrogen digestibility, corrected and uncorrected for uric acid and amino acid digestibility. Amino acid digestibility, as determined by the summation of individual amino acid values, excluding methionine and cystine (81.2%), closely approximated protein digestibility values derived from nitrogen corrected for uric acid (81.4%). Uncorrected nitrogen values gave misleading results (38.4%). Apparent protein digestibility values, when corrected for uric acid, appear to provide a simple and reliable method for estimating protein digestibility.