Evaluation of mycotoxin-contaminated cereals for their use in animal feeds in Hungary.

In the period between December 5, 1991 and September 17, 1998, 760 maize, 367 wheat, 119 soybean, 222 barley, 85 bran, 32 triticale, 60 oat, 14 rye and 22 sunflower samples were investigated for the presence and concentration of seven fusariotoxins (T-2 toxin, zearalenone, deoxynivalenol, nivalenol, diacetoxyscirpenol, HT-2 toxin, fusarenone-X) and OTA. The comparison of analytical data with those of the relevant literature revealed that although the incidence rate and/or concentration of Fusarium mycotoxins and OTA in Hungarian-grown cereals is occasionally considerable, the position of the country is not worse that the average of countries. Our findings indicate that soybean tends to be good substrate for trichothecene-producing fungi and the rate of contamination is regarded as substantial. The commodities were assorted into one of three quality categories. The proportion of objectionable samples was only 3.0, 2.2, 2.3 and 1.7% in maize, wheat, barley and soybean samples, respectively. However, this low rate of objection might still be a source of great economic loss. The proportion of objectionable samples was much higher in the case of bran, oat and triticale (7.1, 6.7, and 6.3%, respectively). The results of the present investigation indicate a need for regular screening for mycotoxins of importance and individual appraisal of each commodity from the point of their use in animal feeds.

Effect of dietary T-2 fusariotoxin concentrations on the health and production of white Pekin duck broilers.

The effects of different dietary levels of T-2 toxin on production, biological, immunological, and pathological parameters of growing white Pekin ducks were studied to establish the "no effect" dietary concentration of, and "no effect" exposure time to, pure T-2 toxin. Day-old white Pekin ducks were randomly allotted to nine groups of 10 ducks each. One group served as a control, and no mycotoxin was added to its feed. The feeds of the experimental groups were supplemented with 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, and 4.0 mg purified T-2 toxin/kg, respectively, from Day 1 until Day 49 of the experiment. Dermatotoxic oral lesions developed in most experimental ducks within 2 d after the start of feeding T-2 toxin-contaminated feeds. The gradual disappearance of macroscopic signs indicated the development of tolerance in groups treated with the lower T-2 toxin content. No repair was found in the 3 and 4 mg/kg groups. Dietary concentrations of T-2 toxin below 0.4 mg/kg had no effect on the average weekly weight gain in the first 6 wk, but a severe decrease was found in the last week of the experiment. The 0.6 mg/kg dietary T-2 toxin had no effect on weight gain in the first 3 wk. At Week 4 and later, the weekly weight gain was significantly reduced, and the final live weight of this group was also significantly lower than that of the control. Dietary T-2 concentrations of 1 mg/kg and greater uniformly depressed growth rate. Only the 3 and 4 mg/kg groups refused feed during the first week. From Week 3 on, the feed intakes of the 0.6 to 4 mg/kg groups were usually less than that of the control group, indicating feed refusal. Serum and plasma chemical values and hematological parameters failed to show dose-dependent effects. The blastogenic response of lymphocytes to nonspecific and specific mitogens was distinctly impaired by the T-2 toxin at all levels in the feed. In the 3 and 4 mg/kg groups, the histological examination revealed lymphocyte depletion in the spleen and bursa of Fabricius.

Effect of various levels of T-2 toxin on the clinical status, performance and metabolism of growing pigs.

In two sets of experiments eight groups of seven-week-old pigs weighing about 9 kg were fed for three weeks a prestarter that contained 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0 or 15.0 mg/kg of highly purified T-2 toxin. The feed of the two control groups was free from T-2 toxin. Average daily intakes of toxin by the pigs were 0.38, 0.81, 1.24, 1.43, 0.93, 0.81, 0.99 and 2.5 mg, respectively. The weight gains, the feed intakes, the extent of feed refusal, the parameters of energy and protein metabolism and the serum concentrations of calcium, inorganic phosphorus and magnesium were affected to different extents by the different doses of T-2 toxin, but the data indicated that feed consumption was reduced and the activity of aspartate aminotransferase was increased by the smallest amount of T-2 toxin tested.

Effect of various levels of T-2 toxin in the immune system of growing pigs.

Four groups of seven-week-old pigs weighing about 9 kg were fed for three weeks a prestarter that contained 0.5, 1.0, 2.0 or 3.0 mg/kg of highly purified T-2 toxin. The average daily intakes of toxin by the pigs were 0.38, 0.81, 1.24 and 1.43 mg, respectively. The experimental and control pigs were immunised with 5 ml aluminum hydroxide gel-absorbed purified horse globulin on the first and fourth days of the treatment period. Blood samples were withdrawn on days 7, 14 and 21 and used for the determination of the titre of anti-horse globulin antibody, for an in vitro lymphocyte proliferation test, using purified horse globulin, phytohaemagglutinin and concanavalin-A and for determinations of the immune complex, the cytotoxic reaction and the phagocytic activity and phagocytic index of circulating granulocytes. The samples taken on day 21 were also used to determine the erythrocyte count, the mean cell volume of the erythrocytes, the haematocrit, the blood haemoglobin concentration, the leucocyte count and the proportion of T lymphocytes. At the end of the experiment samples were taken from the thymus, spleen and mesenteric lymph nodes for histological examination. The diets that contained 2 and 3 mg T-2 toxin/kg caused a significant decrease in the red blood cell count, the mean corpuscular volume and the haemoglobin concentration. A significant decrease in the leucocyte count and the proportion of T lymphocytes was observed in all the treatment groups. There were also dose-dependent, significant decreases in antibody formation and in the blastogenic transformation of lymphocytes, and mild to moderate reactive processes were observed histologically in the lymphoid organs.

Detection of ochratoxin a in human blood and colostrum.

Ochratoxin A (OA) is one of the most frequent sources of mycotoxin contamination of feed plants in Hungary. It is produced by 10% of Aspergillus and 12% of Penicillium species, i.e. by the widely occurring "commonest" mould species. Human exposure to mycotoxins closely resembles that of swine. Fifty-two out of 100 human blood samples collected at random (52%) were found to contain ochratoxin A (0.2-12.9 ng/ml). Recent studies have clearly shown that OA has mutagenic, teratogenic and carcinogenic effects. Sensitivity to mycotoxins is known to be inversely related to age. Therefore, it was considered important to test human colostrum samples for OA content. Thirty-eight out of 92 colostrum samples (41.3%) collected from women in the first 24 hours post partum contained ochratoxin A (0.2-7.3 ng/ml). The HPLC method applied in this study is described in detail.

Effects of T-2 toxin treatment on the egg yield and hatchability in geese.

Ten groups of one-year-old geese, each including 10 layers and 3 ganders, were treated with T-2 toxin at doses of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 and 3.0 mg/kg body weight/day for 18 days by injection into the stomach. At the toxin dose of 0.1 mg/kg/day, the egg yield remained unchanged while the hatching rate slightly decreased. T-2 toxin administered at a dose of 0.2 mg/kg/day decreased the hatching rate and the egg yield. The toxin dose of 0.3 mg/kg/day resulted in a 50% reduction of the hatching rate. The mortality rate was significant at toxin levels higher than 0.8 mg/bwkg/day; however, 30% of the birds survived even the 3.0 mg/kg/day toxin dose.

Perinatal oestrogen syndrome in swine.

Perinatal oestrogen syndrome (F-2 fusariotoxicosis occurring at the perinatal age) was studied in large pig herds and in animal experiments. The disease markedly lowered the conception rate of sows and gilts, and increased the number of repeat breeders. Litter size decreased and the number of stillbirths rose. Both the newborn piglets and the stillborn fetuses showed swelling of the vulva and teats and oedematous infiltration of the perineal region, ventral part of the abdomen and umbilicus, often accompanied by exudative-crusted inflammation, then necrosis of the teats. The number of piglets with splayleg and trembling increased. Gross and histopathological examination revealed enlargement of the ovary and uterus, with signs of follicle maturation in the ovary, glandular proliferation in the endometrium and epithelial proliferation in the vagina in addition to oedema and hyperaemia. In newborn piglets, the signs of hyperoestrogenism could be induced also experimentally, by feeding an F-2 toxin containing diet to pregnant sows. Intrauterine toxin effect was found to be primarily responsible for inducing the syndrome in newborn piglets. Because of its lower quantity, F-2 toxin excreted in the sow's milk is assumed to have a secondary role.

Pathomorphological changes caused by T-2 trichothecene fusariotoxin in geese.

T-2 trichothecene fusariotoxin was administered to 110 laying geese and 33 ganders in the active egg production period through a tube at 2-day intervals on a total of 10 occasions. After the treatment, the geese were subjected to detailed pathomorphological examination. In the ovaries of layers, a cessation of follicle maturation and follicle degeneration dependent on the toxin dose were observed, accompanied by ovulation and consequent peritonitis in the birds that died and in some of those killed by bleeding. Additional findings included involution of the oviduct, lymphocyte depletion, necrosis and amyloidosis in the spleen, catarrhal enteritis, signs of colloid stasis in the thyroid and large numbers of secretory granules in the cytoplasm of the adrenaline-producing cells of the adrenal gland. In the ganders, toxin administration did not reduce the intensity of spermatogenesis but in the spleen, intestine and endocrine glands it caused changes similar to those seen in the layers.

Interaction of T-2 fusariotoxin with anticoccidial efficacy of lasalocid in chickens.

Two battery tests were carried out to reveal whether or not there is an interaction between T-2 fusariotoxin and lasalocid, similar to that known with some ionophorous anticoccidials. In the first experiment, an excessive dose level of toxin (6 ppm) significantly reduced the anticoccidial efficacy of lasalocid at 75 ppm and almost completely eliminated that at 37.5 ppm in cockerels which had been infected with oocysts of Eimeria tenella and E. mitis. In the second experiment, the same dose of lasalocid was tested in combination with 0.5-1.25 ppm levels of toxin, i.e. levels occurring in the field, and all but the lowest level (0.5 ppm) significantly reduced the anticoccidial efficacy of lasalocid. Thus, in cases of outbreaks of coccidiosis, there is need to keep in mind such interaction between anticoccidials and mycotoxins.

Effect of T-2 toxin on egg production and hatchability in laying hens.

The effect of diets containing different levels of T-2 toxin on egg production and hatchability was studied in a four-week experiment using 100 laying hens of the SSL hybrid line and 10 cocks divided into 10 groups. Another aim of the experiment was to investigate how effectively the increased dietary vitamin E content neutralized the adverse effects of T-2 toxin. The diet of the control group (C) contained no mycotoxin, while those of the experimental groups included the following levels of T-2 toxin: groups 1, 2 and 3: 1 mg/kg, groups 4, 5 and 6: 5 mg/kg; groups 7, 8 and 9: 10 mg/kg. Vitamin E was added to the diet of groups C, 1, 4 and 7 at a rate of 50 mg/kg while to that of groups 2, 5 and 8 at a rate of 100 mg/kg. To the diet of groups 3, 6 and 9 no vitamin E was added. Contamination of the diet with T-2 toxin markedly decreased egg production and impaired hatchability. The production decrease was proportional to the T-2 toxin concentration of the diet. Increased dietary vitamin E concentration exerted no influence on egg production. However, during the first week of the experiment it significantly (P < 0.01) decreased the number of infertile eggs and significantly (P < 0.01) improved the hatching percentage. Dietary vitamin E concentration was in positive correlation with the hatching percentage; this correlation was rather close (r = 0.74) in the first week of the experiment.

Subacute toxicity testing of ochratoxin A and citrinin in swine.

Fourteen pigs were fed ochratoxin A and citrinin through a stomach tube at daily doses of 0.02 and 0.01 mg/kg body mass for 57 days. These toxin doses correspond to the average toxin contamination level of feeds in Central Europe. The clinical status of the pigs was monitored and clinical laboratory, haematological and mycotoxin-analytical examinations were performed throughout the trial. At the end of the experiment gross and histopathological examinations were carried out. The results of ochratoxin A and citrinin determination in the blood, obtained by high-performance liquid chromatography (HPLC), are important from the food hygienic point of view. The sensitivity of the method was 2 and 10 ng/ml for ochratoxin A and citrinin, respectively. The recovery rate of the mycotoxins was above 60%.

Changes induced in newborn piglets by the trichothecene toxin T-2.

Three pregnant sows, being in the last quarter of gestation, were used in an experiment to study the changes induced in newborn piglets by T-2 toxin. One sow was used as control (C). The other two received 24 mg (sow A) and 6 mg (sow B) T-2 toxin, respectively, mixed in the feed, daily, up to the time of farrowing. The piglets of sow A became ill by 48-72 h after birth, while the litters of sow B and C remained healthy. The clinical symptoms included faintness, diarrhoea, decreased blood glucose level, and collapse followed by death. The milk and urine of sow A and the stomach contents of affected and dead piglets contained T-2 toxin and its metabolites. Pathological changes seen at necropsy included acute enteritis, degeneration of the liver and kidneys, and oedema of the mesentery. The stomach was filled with clotted milk. Histopathological and electron-microscopic findings consisted of reduced glycogen content and pathological simple fatty infiltration of the liver cells, lymphocyte depletion and necrosis in the lymphoid follicles of the intestinal mucosa, atrophy of the thymic cortex, and hyperfunction of the adrenal and thyroid glands compared to the control.

Mycotoxin research in the Hungarian Central Veterinary Institute.

Mycotoxin research conducted in the Hungarian Central Veterinary Institute is reviewed briefly. The effect of zearalenone was studied by experiments in several animal species (cattle, pig, sheep, chicken, goose, duck, guinea fowl, fish) and in cell cultures in vitro. The chicken (Gallus domesticus) has proved to be resistant to the toxin. In the susceptible species zearalenone causes the most severe damage to the sexual organs. The metabolism of zearalenone, T-2 toxin and diacetoxyscirpenol was also studied. These toxins are resistant to physicochemical factors but are easily transformed in biologically active environment. The acute toxicity study of trichothecene toxins is described along with morphological changes, with particular respect to those of the lymphoid and myeloid organs. Trichothecene toxins impair the natural defence mechanism of the organism and result in the manifestation of different diseases (swine dysentery, caecal coccidiosis). Deoxynivalenol contamination of the feeds, first of all of wheat, is common in Hungary. Its effect on pig fattening was investigated in a field trial. The toxins of different storehouse moulds were also examined.

Effect of T-2 toxin on feed intake, digestion and pathology of rabbits.

Feed containing sublethal T-2 toxin concentrations (12.5 and 25 ppm) was fed to adult rabbits. The animals ate 60-70% less toxin-containing food. The dry matter content of their feces decreased significantly (on an average by 10%). The nutrient digestibility of the feed containing 12.5 ppm T-2 toxin, was increased by 2-6% and that of the 25 ppm T-2 toxin level decreased by 4-11% as compared to the control values. The rabbits showed emaciation, subacute catarrhal gastritis, necrosis of the lymphoid cells of the intestinal mucosa, depletion and necrosis in the lymphoid follicles of the ampulla ilei, spleen and lymph nodes. Necrosis of the cells of mononuclear phagocyte system and myeloid hemacytogenesis was characteristic. The toxin concentration of feces, cecotroph and urine was proportional to intake.

Interaction of T-2 fusariotoxin and monensin in broiler chickens infected with Coccidia.

Field observations suggest that coccidiosis is a common cause of death in broiler chicken flocks fed diets containing sufficient amounts of ionophore antibiotics (monensin, narasin, etc.) and contaminated with mycotoxins, particularly with T-2 fusariotoxin. To study this phenomenon, broiler chickens fed diets containing different amounts of T-2 toxin and free from monensin, or containing a preventive dose (100 mg/kg of feed) of monensin, were infected experimentally with coccidian oocysts. In all groups fed a diet containing monensin plus T-2 toxin severe clinical symptoms of coccidiosis (blood-stained faeces etc). occurred. Deaths and retarded growth depended on the toxin dose and were considerable. The body mass gain of chicks fed a diet containing monensin and T-2 toxin but not infected with coccidia was inferior to that of groups fed diets which contained either monensin or T-2 toxin (experiment 2). On the basis of these findings a negative interaction of the two compounds is assumed. This seems to be supported by the results of experiment 3, i. e. the finding that the lethal dose of narasin, a compound closely related to monensin both in chemical structure and mechanism of action, proved to be much lower (LD50 = 102 mg/kg body mass) for chickens fed a diet supplemented with T-2 toxin than for the control chickens (LD50 = 176 mg/kg body mass). The present results suggest that the feeding of diets severely contaminated with T-2 toxin may alter the anticoccidial efficacy of monensin.

Acute toxicological experiment of T-2 toxin in rabbits.

Rabbits were treated with a single oral dose (1, 2, 4, 6, 8, 10 or 15 mg/kg body mass) of T-2 fusariotoxin. Doses of 4 mg or higher killed the animals in 24 to 48 h. As opposed to the controls, in the treated rabbits gross pathological and histopathological examinations revealed acute catarrhal gastroenteritis, necrosis of lymphoid cells of the gastrointestinal mucosa, centrolobular dystrophy of the liver, necrosis of cells of the mononuclear phagocyte system (MPS) in the liver, tubulonephrosis, focal dystrophy of the adrenal cortex, lymphocyte depletion involving both T- and B-cell-dependent zones of the lymphoid organs (spleen, lymph, ampulla ilei), and depletion and necrosis of the myelopoietic cell colonies of the bone marrow. Similar but milder changes were observed in surviving rabbits exsanguinated 48 h after treatment. In addition to the direct damage done to the digestive tract mucosa and liver, the toxin severely damaged the cells participating in humoral and cell-mediated immunity and in the local defence of the intestinal mucosa, and markedly impaired phagocytosis and granulocytopoiesis. In another experiment rabbits were given oral doses of 2 mg/kg body mass T-2 toxin daily for several days. One rabbit was killed by bleeding every day. In rabbits killed beyond day 7 there was subacute catarrhal gastritis, emaciation, and hypertrophy of the adrenal cortex.