Mycotoxin interactions in poultry and swine

Mycotoxins are toxic compounds produced by fungi. When one mycotoxin is detected, one should suspect that others also are present in a contaminated feed ingredient or finished feeds. The toxicity and clinical signs of observed in animals when more than one mycotoxin is present in feed are complex and diverse. Some mycotoxins, such as the combination of aflatoxin with either ochratoxin A or T-2 toxin, interact to produce synergistic toxicity in broiler chicks. The effects observed during multiple mycotoxin exposure can differ greatly from the effects observed in animals exposed to a single mycotoxin. For example, fatty livers in poultry are used for presumptive diagnostic identification of aflatoxicosis. However, simultaneous presence of ochratoxin A prevents fatty livers. Of the mycotoxin combinations that have been investigated in poultry and swine, the aflatoxin + ochratoxin A and aflatoxin + T-2 toxin interactions appear to be the most toxic.     

日粮添加不同水平的奶牛专用霉菌毒素吸附剂对粪中双歧杆菌数量和乳中黄曲霉毒素含量的影响

奶牛饲料原料中霉菌毒素广泛存在,并严重地威胁着奶牛健康,影响着奶牛生产性能的发挥。本试验选择年龄、胎次、产奶量、泌乳天数相近的荷斯坦奶牛80头探讨日粮添加4种剂量（0g、10g、15g、20g）的霉菌毒素吸附剂对奶牛粪便中双歧杆菌数量和原料奶中黄曲霉毒素残留量的影响。与对照组（添加霉菌毒素吸附剂0g）相比,实验组奶牛粪便中双歧杆菌的外排量明显降低,但各实验组之间统计差异并不显著,仅有数值上的变化。同时,随着霉菌毒素吸附剂含量的增加,原料奶中体细胞数呈现下降的趋势。与试验前相比,原料奶中黄曲霉毒素含量显著降低（P〈0.05）,且随着添加剂量的增加其黄曲霉毒素残留量更少,但添加20g与添加15g相比并无明显变化。研究表明,随着日粮中霉菌毒素吸附剂不同量的添加,均可有效降低原料奶中黄曲霉毒素的残留量,进而提高原料奶质量。     

Pathological studies in broiler chicks fed aflatoxin or ochratoxin and inoculated with inclusion body hepatitis virus singly and in concurrence

Day-old broiler chicks found negative for maternal antibodies against inclusion body hepatitis (IBH) virus by agar gel precipitation test and viral antigen in cloacal swabs by dot enzyme immunoassay were divided into 6 groups of 20 chicks each. Group A was fed aflatoxin B₁ at 1.25 ppm from 3 to 38 days of age; group O was fed ochratoxin A at 0.5 ppm from 3 to 38 days of age; group V was inoculated with 1 ml of IBH virus of titre log₁₀ 6.5 EID₅₀ per 0.2 ml. Groups AV and OV were given aflatoxin B₁ and ochratoxin A, respectively, and also infected with the virus. Group C served as control. There was mild enlargement and paleness of the liver up to 18 days post inoculation in group V; there were no lesions in group A; and there was gradual enlargement of the kidneys from 10 days post feeding of mycotoxin onwards in group O. In the combined groups AV and OV the gross lesions were slightly more severe. In group V, varying degrees of degenerative histopathological changes, congestion and haemorrhages were seen particularly in the liver, followed by the kidneys, bursa, spleen, myocardium and lungs, along with intranuclear inclusion bodies in the hepatocytes, mostly in the early stages of infection. Similar microscopic changes, but without inclusion bodies, were seen in groups A and O and the changes were pronounced in the later stages. In group O, the kidney lesions were more pronounced than the liver lesions. In the concurrently infected groups, AV and OV, the changes were similar but slightly more marked than in the corresponding individual groups. Inclusion bodies in hepatocytes were more frequent, more prominent and appeared earlier in the concurrent groups.Abbreviations AGPT agar gel precipitation test - DPF days post feeding of the mycotoxin - DPI days post inoculation - EIA enzyme immunoassay - EID₅₀ dose at which 50% of embryos will be infected - IBH inclusion body hepatitis - PAU Punjab Agricultural University     

日粮添加不同水平的奶牛专用霉菌毒素吸附剂对粪中双歧杆菌数量和乳中黄曲霉毒素含量的影响

奶牛饲料原料中霉菌毒素广泛存在,并严重地威胁着奶牛健康,影响着奶牛生产性能的发挥。本试验选择年龄、胎次、产奶量、泌乳天数相近的荷斯坦牛80头探讨日粮添加4种剂量（0、10、15、20g）的霉菌毒素吸附剂对奶牛粪便中双歧杆菌数量和原料奶中黄曲霉毒素残留量的影响。与对照组（添加霉菌毒素吸附剂0g）相比,试验组奶牛粪便中双歧杆菌的外排量明显降低,但各实验组之间差异不显著,仅有数值上的变化。同时,随着霉菌毒素吸附剂含量的增加,原料奶中体细胞数呈现下降的趋势。与试验前相比,原料奶中黄曲霉毒素含量显著降低（P〈0.05）,且随着添加剂量的增加其黄曲霉毒素残留量更少,但添加20g与15g间无明显变化。因此,本研究表明,随着日粮中霉菌毒素吸附剂添加量的增加,可有效降低原料奶中黄曲霉毒素的残留量,进而提高原料奶质量。     

Decreased egg production in commercial laying hens fed zearalenone- and deoxynivalenol-contaminated grain sorghum

In an experiment to determine the effect of corn vs. grain sorghum on performance of laying hens, egg production decreased significantly in the grain sorghum-fed hens in each of two trials starting 24 weeks after the trials began. Necropsy of chickens fed both diets revealed buccal ulceration at the ventral aspect of the oral cavity and squamous metaplasia of the esophageal glands and submaxillary salivary glands. Lesions were much more severe in the sorghum-fed birds than in the corn-fed birds. Analysis of the grain sorghum and corn revealed the presence of mycotoxins. Zearalenone and deoxynivalenol were present in the grain sorghum, and a lower amount of deoxynivalenol and a trace of aflatoxin B1 were found in the corn. Although mycotoxin levels were low, interaction between these mycotoxins and others may have decreased egg production.     

Evaluation of two mycotoxin binders to reduce toxicity of broiler diets containing ochratoxin A and T-2 toxin contaminated grain

In order to assess ochratoxin A (OA) and T-2 toxin (T-2) binding ability of two commercial sorbents, both in vitro and in vivo trials with broilers were performed. Crude OA and T-2 extracts from contaminated grain were used to assess in vitro binding ability of two sorbents (Zeotek [Zk] and Mycofix [Mx]), by quantifying free mycotoxin through an enzyme-linked immunosorbent assay (ELISA) test. For in vivo trial, a 3 x 2 x 2 factorial arrangement was used for this experiment, being the factors: adsorbents (none, Zk, and Mx), OA (0 and 567 parts per billion [ppb]) and T-2 (0 and 927 ppb). OA and T-2 contaminated wheat and corn, respectively, were added to sorghum-soybean meal diets to meet 567 ppb of OA and 927 ppb of T-2. Mycotoxins were fed alone or combined in treatments. After 21 days, blood chemistry, gross, and histological evaluations were performed. Relative weights of liver, kidney, and bursa of Fabricius were obtained. Zk had the highest OA and T-2 in vitro binding ability (100% and 8.67%, respectively). Chickens fed OA with or without sorbents had a lower body weight and feed intake reduction. However, those birds fed T-2 were partly protected by a sorbent. Birds fed both toxins showed toxic additive effects, and no protection of any adsorbent was observed. A significant reduction in plasma proteins, albumin, and globulins was a characteristic observed in all birds fed diets with OA both with or without adsorbents. Uric acid level in blood was increased in all chickens fed OA-contaminated diets. Histological findings observed in birds fed OA-contaminated diets were necrosis of kidney tubular cells, swollen and necrotic hepatocytes, bile ducts hyperplasia, and increased diameter of proventriculus glands. In birds that received T-2 alone, only the liver, with the same kind of lesions, was affected. According to these results, it can be concluded that there is not a relation between in vitro and in vivo trials. OA toxic effects could not be counteracted by any sorbent. T-2 toxicity could be partially counteracted by an adsorbent used in this research.     

Clinico-pathomorphological, serum biochemical and histological studies in broilers fed ochratoxin A and a toxin deactivator (Mycofix Plus)

1. Toxic effects of two concentrations (0.5 and 1 mg/kg) of ochratoxin A (OTA) and attenuating effects of a toxin deactivator (Mycofix Plus(MTV INSIDE)) containing the yeast Trichosporon mycotoxinivorans on the performance (feed conversion ratio; body weight gain), serum enzymes (lactate dehydrogenase, gamma-glutamyltranspeptidase and aspartate aminotransferase) and clinico-pathomorphology of internal organs were studied in 270 one-day-old broiler chicks divided into 9 groups over a 42-d period. 2. Feed conversion ratios (FCR) in groups fed toxin deactivator were improved compared with groups receiving OTA only. An increase in the relative weight of kidney and liver was observed in groups fed 0.5 and 1 mg/kg OTA on day 42 of the experiment as compared with the control group. In contrast, relative weights of bursa of Fabricius and spleen were not significantly affected in experimental groups exposed to OTA as compared to control groups determined on days 28 and 42 of age. 3. Serum enzymes (LDH, GGT and AST) values in OTA treated groups determined on days 28 and 42 were higher than those of the control group. 4. Histopathological examination of kidney on day 42 revealed degenerative changes in the epithelial cells of the proximal convoluted tubules and massive necrosis of the proximal tubular epithelial cells. These changes were less marked in birds receiving 0.5 mg/kg OTA than in those receiving 1 mg/kg. In general, histological changes in kidneys, liver, bursa and spleen were less pronounced in birds receiving OTA and toxin deactivator concomitantly. 5. Dietary OTA at 0.5 and 1 mg/kg adversely affects FCR, increases the serum liver enzymes and induces pronounced pathomorphological and histological changes in internal organs of broiler chicks. Co-administration of OTA with deactivator attenuated the harmful effects.     

Production, hatchability and fertility of eggs from breeding Japanese quail (Coturnix coturnix japonica) fed diets containing furazolidone.

1. Breeding Japanese quail were allocated to 8 groups, each group consisting of 20 females and males. The birds were fed one of 4 diets for up to 33 d: a control diet or a diet containing 200 mg/kg, 400 mg/kg or 1000 mg/kg furazolidone. Subsequently, quails were fed a furazolidone-free diet for up to 21 d. Egg production, quality, hatchability and fertility of the groups were measured. 2. Significant reduction in egg production occurred in birds fed 400 mg/kg and 1000 mg/kg furazolidone, the effect being more pronounced at the higher concentration. 3. Hatchability was reduced significantly for all groups of birds fed furazolidone and this effect was both dose and time dependent. The reduction in hatchability was attributable to an increase in infertile eggs rather than an increase in embryonic mortality. 4. Egg quality was affected, with more small eggs being produced by birds fed 1000 mg/kg furazolidone. 5. After removal of the experimental diets egg production of the affected groups returned to control values. Hatchability and fertility of affected groups also returned toward control values, but had generally not attained these values 21 d after the cessation of the experimental diets. 6. It was concluded that standard recommendations for the therapeutic dosage of poultry with furazolidone may not be appropriate for breeding Japanese quail.     

Preparation of the mycotoxin ochratoxin A for research and diagnostic purposes

The following fungal strains were tested for the production of the mycotoxin ochratoxin A: Aspergillus melleus (CCM F 802), Aspergillus ochraceus (CCM 8002) and Aspergillus ochraceus (CCM F 803). The strain Aspergillus melleus (CCM F 802) proved to be very suitable for the laboratory preparation of ochratoxin A. The mould was multiplied on a natural substrate for 14 days at the temperature of 28 degrees C and at 100% relative humidity. Ochratoxin A was extracted into organic solvents (chloroform + acetic acid), purified with aqueous alkaline solutions and by preparative thin-layer chromatography. The isolated mycotoxin was identified by physico-chemical methods. The amount of pure ochratoxin A isolated from 600 g of substrate was 305 mg.     

Sequential studies of skeletal calcium reserves and structural bone volume in a commercial layer flock.

1. Production-induced osteoporosis in caged laying hens is thought to represent a major constraint to continued genetic development. 2. The relationship between body weight, egg production, skeletal abnormalities characteristic of osteoporosis, femur calcium and bone histology was examined in a flock of ISA Brown layers from 16 to 68 weeks of age. 3. Experiment 2 examined a flock of Lohmann browns for skeletal abnormalities characteristic of osteoporosis at 45 weeks of age and the severity of abnormalities was then related to body weight and production between 18 and 45 weeks of age. 4. Average body weight declined in the ISA flock between 35 and 45 weeks of age, which correlated with a loss of skeletal calcium reserves (15% to 20%) and with the induction of osteoporosis. Between 42 and 68 weeks of age, birds were able to replenish femur calcium levels. 5. Birds in the Lohmann flock showing severe skeletal abnormalities at 45 weeks of age experienced weight loss between 27 and 31 weeks of age, which was associated with a decrease in egg production of 18%. After 35 weeks of age, egg production of these birds recovered to similar levels as unaffected or mildly affected birds. 6. It seems likely that better standardisation of the equilibrium between growth, skeletal reserves, food intake and egg production can reduce osteoporosis, as well as improving the productive potential of modern laying strains.     

Experimental combined aflatoxin B1 and ochratoxin A intoxication in pigs

Twenty-one pigs weighing approximately 18 kg were placed in 7 groups of 3 and given diets containing respectively aflatoxin B1 alone at 0.375 and 0.0750 mg/kg, ochratoxin A alone at 1 and 2 mg/kg, 0.375 mg/kg of aflatoxin B1 plus 1 mg/kg of ochratoxin A and 0.750 mg/kg aflatoxin B1 and 2 mg/kg of ochratoxin A. The remaining group served as untreated control. At the respective dose levels, pigs receiving similar doses of ochratoxin A alone or in combination with aflatoxin B1, were similarly affected, the clinical effects of aflatoxin having been mostly obscured by those due to ochratoxin A. Mild degenerative hepatic changes typical of aflatoxicosis were observed in pigs fed this toxin alone or in combination with ochratoxin A. In kidneys of pigs fed diet containing 1 and 2 mg of ochratoxin A alone changes included interstitial fibrosis of the vortex and dystrophy and degeneration of the tubular epithelium. Similar lesions but less pronounced fibrosis were found in kidneys of pigs receiving both toxins. The respective lower dose levels of mycotoxins selected were judged to be about the no-effect levels for each dosed separately under the conditions of the trial. Such levels have been found not infrequently on mould affected grain and stock foods. The result highlights the difficulties that may be experienced in the recognition of such multimycotoxicoses as they are likely to occur in the field and indicate the need for toxicological analysis as well as pathological investigation in establishing a diagnosis.     

Effects of mould and toxin contaminated barley on laying hens performance and health.

Moulded and mycotoxin containing barley was incorporated into the diets for laying hens to study the effects on performance and health. Health indicators were different blood plasma parameters and liver vitamin A and E levels. A total of 30 hens were fed 3 diets, one supplemented with 30% of toxin-free and two with differently moulded barley from 1997 and 1998 for 7 weeks. The moulded diets contained low to moderate concentrations of ochratoxin A, zearalenone, deoxynivalenol and nivalenol. Inclusion of mouldy barley in the diets had an adverse effect on feed intake, feed conversion, digestibility of nutrients, egg production and egg quality. Plasma alkaline phosphatase was increased and certain biochemical blood parameters (bilirubin, uric acid, chlorine, protein, albumin, vitamin A) were also higher or changed compared to control. The ochratoxin A contamination although relatively low could have contributed to some of these effects as well as reduced intake of feed. The higher mould contamination and an unidentified cell-toxic constituent in the diet containing barley from 1998 can probably also explain the more marked effects from this diet.     

霉菌毒素吸附剂对产蛋鸡生产性能、蛋黄中微量元素含量、血清抗氧化和生化指标的影响

本试验旨在研究霉菌毒素吸附剂(分别命名为CZ和SF)对产蛋鸡生产性能、蛋黄中微量元素含量、血清抗氧化和生化指标的影响。选取216只38周龄健康的罗曼蛋鸡,随机分成3个组,每组6个重复,每个重复12只。对照组饲喂基础饲粮,试验组(CZ组、SF组)分别在基础饲粮中添加0.5 g/kg的CZ和SF。预试期为10 d,正试期为35 d。结果表明:1)与对照组相比,CZ组产蛋鸡试验3~5周产蛋率、日产蛋量显著提高(P0.05);饲粮中添加霉菌毒素吸附剂对产蛋鸡的平均蛋重、平均日采食量、料蛋比和死淘率均无显著影响(P0.05)。2)与对照组相比,各试验组产蛋鸡第14天和第35天蛋黄铜、锰含量均无显著差异(P0.05);CZ组第14天蛋黄锌含量显著提高(P0.05)。3)饲粮中添加霉菌毒素吸附剂对第14天和第35天产蛋鸡血清谷胱甘肽过氧化物酶活性和丙二醛含量无显著影响(P0.05)。与对照组相比,各试验组产蛋鸡第14天血清总抗氧化能力均显著提高(P0.05);SF组第35天血清总超氧化物歧化酶活性显著提高(P0.05)。4)与对照组相比,CZ组产蛋鸡血清碱性磷酸酶活性、葡萄糖含量显著降低(P0.05);SF组血清总胆固醇含量显著降低(P0.05)。由此可见,饲粮中添加霉菌毒素吸附剂CZ明显提高了产蛋鸡产蛋性能,2种吸附剂CZ和SF都增强了产蛋鸡血清抗氧化功能,改善了部分血清生化指标。     

Ultrastructural study of the liver and kidney in ochratoxicosis A in young broiler chicks

Ultrastructural changes are reported in the kidney and liver of 20-day-old broiler chicks fed ochratoxin A (OA), incorporated in the diet at levels of 2 and 4 ppm. Changes in the kidney included the presence of abnormally shaped mitochondria in the proximal convoluted tubules. There was an increase in the size and number of mitochondrial dense granules and cytoplasmic peroxisomes. Intranuclear and cytoplasmic lipid droplets and electron dense round bodies in the dilated smooth endoplasmic reticulum were also noted. Regional thickening and degeneration of the glomerular basement membrane was observed in some cases. In the liver from OA fed birds there was an increased accumulation of cytoplasmic glycogen in the hepatocytes. Abnormal mitochondrial ring forms in the kidney and the accumulation of glycogen in the liver are considered to be of diagnostic significance in ochratoxicosis of young broiler chicks. The severity of the changes was found to be dose related. These results suggest that the mitochondria in the proximal convoluted tubules of kidney were most sensitive to OA toxicity.     

Immunoenzyme detection of the mycotoxin, ochratoxin A]

Indirect, enzymoimmunological assays of the mycotoxin ochratoxin A were developed. In this technique a polyclonal (rabbit) antibody to ochratoxin A was used, along with the other, peroxidase-labelled (pig anti rabbit) antibody. The sensitivity of this method ranged around 75 pg of ochratoxin A per pit. The range of calibration curve was from 10 to 1000 pg per pit. The cross reactions with other ochratoxins made 1.4% (ochratoxin C). The ELISA test of ochratoxin A can be used as an expeditious screening method for a preliminary examination of the greater number of samples.     

Effect of dietary exposure to aflatoxin B1 on resistance of young chickens to organophosphate pesticide challenge

White leghorn chickens were placed on a diet containing 0, 1000, or 3000 ppb aflatoxin B1 for 7 weeks. At the end of that time, the birds were challenged orally with the organophosphate pesticide malathion. A malathion dose of 215 mg/kg resulted in significant clinical signs of cholinergic poisoning in 4/10, 5/10, and 8/10 birds fed 0, 1000, and 3000 ppb aflatoxin B1, respectively, and the chickens required antidotal atropine 40 min later. Activity of brain cholinesterase was significantly lower than control levels in all birds given this dose of malathion, with activities being 28% +/- 6, 21% +/- 2, and 15% +/- 2 of control values (Mean +/- standard error, N = 5) if fed 0, 1000, and 3000 ppb aflatoxin B1, respectively. Plasma cholinesterase values paralleled those of brain, with significantly more inhibition in samples from birds given 1000 and 3000 ppb aflatoxins with malathion. Brain and plasma cholinesterase activities in birds fed aflatoxins and given a dose of malathion below the threshold for cholinergic signs (125 mg/kg) were also lower than activities in birds given malathion alone. Although aflatoxin alone had no direct effect on the activities of these enzymes, it appears that this mycotoxin may contribute to the esterase inhibition that is a manifestation of the acute toxic effects of malathion in chickens.     

The effects of low doses of Senna occidentalis seeds on broiler chickens

The effects of 0.5%, 0.3% and 0.1% w/w concentrations of Senna occidentalis (So) seed mixed with commercial ration were studied in 18 groups of 32 broiler chicks each, from 1 day to 49 days of age. Three groups were fed one of the rations throughout their lives (TL). Three other groups were fed one of the rations from the 1st to the 28th day of life (starter phase, SP), and the final 3 groups were fed one of the rations from the 29th to 49th day (finisher phase, FP). Each experimental group was matched by a control group fed the same diet over the same period but without the inclusion of So. All the animals were killed at 49 days of age, and blood was collected from 10 birds in each group for biochemical studies (ALT, AST, GGT, LDH, UA). A complete necropsy was performed on 3 birds from each group. No significant differences in the biochemical parameters in the serum were found between the control and experimental chicks, but animals treated with 0.5% So in groups FP and TL, gained less weight and chicks that received 0.3% So or 0.5% So in the ration throughout life (TL) had a larger feed conversion ratio. Besides this, degenerative changes were found in the striated skeletal muscle in the chest, in the myocardium and in the liver in the animals that received the higher concentrations of So seeds.     

Comparison of effects of dietary T-2 toxin on growth, immunogenic organs, antibody formation, and pathologic changes in turkeys and chickens.

The effect of T-2 toxin (trichothecene mycotoxin) consumption on Broad-Breasted White turkey poults and White Leghorn chicks was studied. Groups of ten 8-day-old poults were fed rations containing T-2 at 10 ppm, 2ppm, or 0 ppm (controls) for a period of 4 weeks; a 4th group (inanition control) was fed control rations equal to the amount consumed by the group fed rations containing T-2 at 10 ppm during the previous 24 hours. A similar experimental design was used to study the effect of the toxin on 1-day-old chicks. The thymus glands of the poults given the feed containing 10 ppm were markedly decreased in size compared with thymus glands from poults in the control group, 0.182 vs 0.331 (percentage of body weight). There was no significant (P less than or equal to 0.05) decrease in thymus gland size in poults given 2 ppm or in the inanition controls. Dietary treatment did not appear to affect the size of the bursa or spleen of the poults. Histopathologic examination of thymus glands from poults given 10 ppm of T-2 revealed a depletion of cortical lymphocytes. Chicks appeared less sensitive to T-2 toxin than did the poults. There was no effect by any dietary treatment on the size of the thymus gland, bursa, or spleen of chicks. Reductions were noticed in feed efficiency and weight gain. There was no effect of T-2 toxin on agglutinating antibody formation to Pasteurella multocida bacterin.     

Isolation of ochratoxin A in fodder barley]

The authors describe the finding and identification of ochratoxin A in fodder barley of Czechoslovak produce. The fungus Penicillium verrucosum var. verrucosum, which is an ample producer of ochratoxin A, was isolated from a sample of barley. The isolated fungus was cultivated on several substrates at different temperatures and the production of ochratoxin A was studied during cultivation. The authors believe that the ochratoxicoses of farm animals may also occur in Czechoslovakia and that it would be useful to study this mycotoxin in selected feeds.