Experimental trichothecene mycotoxicosis produced in broiler chickens by Fusarium sporotrichiella var. sporotrichioides

Fusarium sporotrichiella var. sporotrichioides (Bilay), cultured on . sterilised popcorn at 23 degrees C and then at 8 degrees C, 16 degrees C and 23 degrees C and fed as 50% of the diet, was lethal to 7-day-old male broiler chickens. The 8 degrees C culture, containing T-2 toxin at 50 parts per million (ppm) and neosolaniol at 5 ppm, was given as whole culture at dietary concentrations of 10%, 5%, 1% and 0% for 17 days and 1% for 42 days. Half the chickens that were fed the 10% diet died during the 17 days (5 ppm T-2 toxin and 0.5 ppm neosolaniol). The corresponding daily dose was 0.24 mg T-2 toxin and 0.02 mg neosolaniol/kg body weight/day. The chickens that died were dehydrated, had necrosis and depletion of lymphoid and haematopoietic tissues and necrosis of the hepatobiliary system, gastroenteric mucosa, feather epidermis and renal tubular epithelium. The survivors had anaemia, reduction of weight gain and transiently altered righting reflex. The comb and beak were pale yellow and the feather barbs were dishevelled. Survivors also had atrophied lymphoid tissues, reduced haematopoietic cellularity in the bone marrow, necrosis of oral and crop mucosa, vacuolated hepatocytes, hyperplastic bile ductules, and reduction of the thyroid follicular diameter.     

Mycotoxicosis produced in broiler chickens by multiple doses of either t‐2 toxin or diacetoxyscirpenol

T‐2 toxin, a 12, 13‐epoxytrichothecene mycotoxin, was given by crop gavage to 7‐day‐old male broiler chickens as 14 daily doses of 1.5, 2.0, 2.5, and 3.0 mg/kg body weight/day. Diacetoxyscirpenol, also an epoxytrichothecene, was given at doses of 2.5, 3.0,and 3.5 mg/kg/day. Some chickens became dehydrated and emaciated and died. In survivors, body weight and haematocrit were reduced, the feathers were malformed, and the beak and legs were pale yellow. At necropsy, the lymphoid organs were atrophic, bone marrow was pale red or yellow, the liver was discoloured yellow, and the crop mucosa was ulcerated.

Microscopic lesions included necrosis and cell depletion in lymphocytic and haematopoietic tissues, and necrosis of hepatocytes, bile ductule epithelium, enteric mucosa, and germinal regions of feather barbs. Other hepatic lesions were fatty change of hepatocytes and hyperplasia of bile ductules. Thyroid follicles were small, contained pale colloid and had tall epithelial cells. T‐2 toxin was more detrimental than diacetoxyscirpenol to lymphocytic and haematopoietic tissues.     

T-2 toxin effect on rat aorta: cellular changes in vivo and growth of smooth muscle cells in vitro

Rats were injected intraperitoneally with T-2 toxin and their aortas were studied by light and electron microscopy. The growth of smooth muscle cell explants taken from the tunica media of aortas of similarly treated animals was observed. A single large dose (2 mg/kg) or four injections of 0.3 mg/kg T-2 toxin caused damage and occasional necrosis of endothelial cells, accumulation of basement membrane-like material in the intima, and swelling and activation of smooth muscle cells in the tunica media. Three or more weeks after the last injection of 0.3 mg/kg T-2 toxin the endothelial cells were normal but an excess of fragmented intimal basement membrane-like material persisted and smooth muscle cells were still activated. Outgrowths from explants of aortic tunica media taken within 1 week of the last dose of T-2 toxin showed marked inhibition of smooth muscle cell growth. Three or more weeks after the toxin, the explants showed significantly increased outgrowths. These findings suggest that T-2 toxin causes early endothelial and smooth muscle cell injury accompanied by inhibition of smooth muscle cell growth in culture. This is followed by stimulation of the proliferative capacity of smooth muscle cells in vitro. If a similar mechanism is operative in vivo, it could explain the chronic vascular changes observed after limited exposure to T-2 toxin.     

Experimental T-2 toxicosis in swine following inhalation exposure: effects on pulmonary and systemic immunity, and morphologic changes

Thirty-four, 9- to 11-week-old, male castrated, crossbred, specific pathogen-free derived pigs were exposed to a T-2 toxin aerosol at a nebulized dose of 0 or 9 mg/kg in pairs, each pair consisting of 1 control and 1 T-2 treated pig which were exposed on the same day. Twenty to 30% of the toxin (1.8 to 2.7 mg/kg) was retained by the pigs. Five pairs were killed on each of 1, 3 and 7 days after dosing. Two pairs of pigs were designated as a 0.33-day group when one T-2 treated pig died and the other was killed in a moribund state at 8 to 10 hours after dosing. The pulmonary and systemic immunity and morphologic changes of the lungs and other organs were examined. Bronchoalveolar lavage was performed to obtain alveolar macrophages (AM) and pulmonary lymphocytes (PL). The phagocytic ability of AM and mitogen-induced blastogenic responses of enriched PL and peripheral blood lymphocytes were evaluated. Clinically, all of the T-2 treated pigs vomited and were cyanotic, anorexic, lethargic and laterally recumbent. In the 0.33-, 1-, and 3-day T-2 treated pigs, there was a marked reduction in AM phagocytosis and mitogen-induced blastogenic responses of PL but not of peripheral blood lymphocytes. Mild to moderate, multifocal interstitial pneumonia was seen in the majority of the T-2 treated pigs. In pigs dying following inhalation of T-2 toxin, there was a more severe pneumonia, as well as marked necrosis of lymphoid tissues, severe necrohemorrhagic gastroenteritis and edema of the gall bladder wall, and multifocal necrosis of the heart and pancreas. Thus, inhalation exposure to T-2 toxin can result in clinical signs and morphologic changes resembling those reported previously in pigs given T-2 toxin intravascularly (iv) at a dose of 1.2 mg/kg (approximate LD50) or greater, as well as death. Mild pulmonary injury as well as transient impairment of pulmonary immunity was present in pigs surviving inhalation exposure.     

Hepatic necrosis and glutathione depletion in captopril-treated mice.

Captopril (CP) is an angiotensin-converting enzyme inhibitor whose metabolism involves endogenous thiols which may be depleted at high doses of CP. Following intraperitoneal administration of CP (50-300 mg/kg), dose-dependent depletion of hepatic glutathione, increased serum transaminase (SGPT) levels and hepatic necrosis were observed. The hepatic necrosis observed was either subcapsular or parenchymal in distribution. Both types of necrosis showed a dose-dependent increase in severity but with a large inter-animal variation. The patterns of necrosis observed with CP are different from the necrosis caused by paracetamol. Oral CP (300 mg/kg) caused parenchymal necrosis in only one animal. It is suggested that subcapsular necrosis may be due to the direct effect of i.p. captopril whereas parenchymal necrosis may be a consequence of hepatic GSH depletion.     

Hepatic necrosis and glutathione depletion in captopril-treated mice

Captopril (CP) is an angiotensin-converting enzyme inhibitor whose metabolism involves endogenous thiols which may be depleted at high doses of CP. Following intraperitoneal administration of CP (50-300 mg/kg), dose-dependent depletion of hepatic glutathione, increased serum transaminase (SGPT) levels and hepatic necrosis were observed. The hepatic necrosis observed was either subcapsular or parenchymal in distribution. Both types of necrosis showed a dose-dependent increase in severity but with a large inter-animal variation. The patterns of necrosis observed with CP are different from the necrosis caused by paracetamol. Oral CP (300 mg/kg) caused parenchymal necrosis in only one animal. It is suggested that subcapsular necrosis may be due to the direct effect of i.p. captopril whereas parenchymal necrosis may be a consequence of hepatic GSH depletion.     

Changes in free amino acids in Peripheral Blood (PB) lymphocytes and Polymorphonuclear (PMN) leukocytes after treatment with diazepam

The effect of single and chronic (15 days) i.p. injections (1.0 and 8.0 mg/kg) of diazepam (DZ) on free amino acid profile in peripheral blood (PB) lymphocytes and polymorphonuclear (PMN) leukocytes of male Wister Albino rats were investigated. Depletion of some free amino acids was observed in the lymphocytes (mixed T- and B-lymphocytes) and PMN leukocytes (91–95%) neutrophils especially after chronic DZ-treatment. A dose-dependent depletion in the lymphocyte amino acids, Tau, Gly, Ala, Met and Ile, was found after both acute and chronic DZ-treatment. A similar depletion of Tau, Asp, Glu and Met appeared in the PMN leukocytes after single doses as well as chronic DZ-treatment. These results suggest that administration of 1.0 – 8.0 mg/kg of DZ in single dose or after chronic administration may interfere with the transport of certain important amino acids and/or protein turnover in PB lymphocytes and PMN leukocytes.On the other hand, the basic amino acids Lys, His and Arg were significantly increased in PMN leukocytes after chronic administration of 1.0 mg/kg DZ. It was suggested that the increased levels of the basic amino acids in the neutrophils may interact with the intracellular changes in pH that normally accompany the respiratory burst.     

Detection of fusarin C and trichothecenes in Fusarium strains from Spain.

A group of 49 strains of Fusarium sp. isolated from different Spanish samples of cereals and mixed feedstuffs were screened for their ability to produce trichothecenes like T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS) and deoxynivalenol (DN), as well as other mycotoxin produced by Fusarium named fusarin C. The production of these mycotoxins was analyzed by means of spectrophotometry, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography (GC). Results showed that from 19 Fusarium strains in which cultures trichothecene production was detected, 15 were HT-2 producers, 9 T-2 producers, 14 DAS producers and 10 DN producers. On the other hand, from 28 Fusarium strains in which cultures fusarin C production was detected, 22 were low fusarin C producers (ranged from 0.04 to 1 microgram/l ICI medium), 5 Fusarium strains were intermediate-level producers (ranged from 1 to 10 micrograms/l ICI medium) and one Fusarium strain produced 240 micrograms/l ICI medium. The identified Fusarium strains that produced trichothecenes and fusarin C were F. moniliforme and F. oxysporum.     

An efficient method for producing and purifying gramme quantities of T-2 toxin

A simple procedure was developed for the laboratory production and purification of gramme quantities of T-2 toxin. After growing at 22 °C on rice for two weeks, Fusarium sporotrichioides T-21 produced average T-2 toxin concentrations of 1309 mg/kg and as much as 1000 mg of crystalline product were recovered. The method described avoids long-term, low temperature incubations. Crude culture extracts were rapidly purified in two steps by column chromatography on silica gel. The purity of the crystallized product was verified by thin-layer chromatography using various spray reagents for detection.     

The therapeutic potential of thiamine for treatment of experimentally induced subacute lead poisoning in sheep

Lead poisoning remains a serious problem in veterinary and human medicine. A number of lead chelators have been used for treatment of lead intoxication, but none of them is completely effective to remove lead from all organs. Therefore, alternatives for the treatment of lead poisoning are required. In this study, the efficacy of thiamine on blood and tissue lead contents was evaluated in subclinical lead toxicosis in sheep. Nine female sheep weighing 25–29 kg were orally receiving a daily dose of 80 mg/kg body weight of lead acetate for 5 days. Then, the animals were assigned into two groups. Group 1 did not receive any further treatment and was used as the control group, and group 2 was treated intravenously by 25 mg/kg body weight of thiamine twice daily for 7 days. Within 1 day following treatment, in the treated group, blood lead level (mean 243.5 μg/l) was significantly (P < 0.05) lower than that in group 1 (mean, 518.16 μg/l). Thiamine treatment significantly reduced ovary lead content. A significant reduction of serum zinc concentration was also observed in thiamine treated animals. These results suggest that thiamine might have some therapeutic effects on lead poisoning, but the zinc status of depletion should be considered during long periods of treatment.     

Acute toxicity of citrinin in turkeys and ducklings

Citrinin, a naturally occurring mycotoxin, was dissolved in dimethyl-sulphoxide - 70% ethanol (3:1, v/v) and administered orally in two trials to 7-day-old male turkey poults and male white Pekin ducklings. The single dose LD50 value in 7-day-old male turkey poults was 56 mg/kg and in 7-day-old male white Pekin ducklings it was 57 mg/kg. The mycotoxin was nephrotoxic in both species, but the renal lesions were more severe in turkeys and were characterised by degeneration and necrosis of renal tubular epithelium. In turkeys, lesions were found in the liver and included hepatic cell necrosis and biliary hyperplasia. Lymphoid necrosis with depletion involved the thymus and cloacal bursa of turkeys and ducklings. These latter lesions were the most prominent histopathological alterations in citrinin-treated ducklings.     

Taishan Pinus massoniana Pollen Polysaccharides Enhance Immune Responses in Chickens Infected by Avian Leukosis Virus Subgroup B

Immunosuppressive virus, which can cause suppressed immunity and vaccination failure, frequently occurs in chicken flocks and seriously destroys the poultry industry. Our previous studies have reported that Taishan Pinus massoniana pollen polysaccharide (TPPPS) possess immunomodulatory effects and improve the immune effects of vaccines. In this study, avian leukosis virus subgroup B (ALV-B) was chosen as immunosuppressive virus to artificially establish immunosuppressive models in chickens, and the immune modulatory ability of TPPPS on the immune response of chickens was evaluated. Four randomly assigned groups (Group I–IV) of these immunosuppressed chickens were administered with TPPPS at doses of 0, 100, 200, and 400 mg/kg (every kilogram chick), respectively. Group V was administered with saline as control. At seven day old, 10 chickens randomly selected from Group I–V were inoculated with the attenuated Newcastle disease (ND) vaccine. The results showed that during the monitoring period, TPPPS significantly enhanced weight of immune organs, peripheral lymphocyte proliferation, the percentage of CD4+ and the ratio of CD4+/CD8+, IL-2 and IFN-γ production, and ALV-B antibody positive rate of chickens in a dose-dependent manner, with 400 mg/kg TPPPS being the most effective. In addition, the antibody titer against Newcastle disease virus (NDV) in Group IV with 400 mg/kg was significantly higher than those in other groups. We observed the stronger immunity in the TPPPS group, which indicates that TPPPS could be used as an immunoenhancer to relieve immunosuppression caused by ALV-B in the poultry industry.     

Apparent analgesic effects of morphine in chickens may be confounded by motor deficits

Three experiments were conducted with chickens to examine the effects of morphine on nociception and motor coordination. In Experiment 1, using shock-elicited vocalization as an index of pain, doses of 5, 20, and 30 mg/kg of morphine failed to affect vocalization thresholds. In the second experiment, 30 mg/kg of morphine failed to affect vocalization thresholds at varying times since injection. In Experiment 3, 30 mg/kg of morphine significantly impaired movement in response to nonaversive stimulation. These results show that previous evidence for an analgesic effect of morphine in chickens may have been due to morphine effects on motor initiation and/or coordination.     

The cognitive enhancer T-588 partially compensates the motor associative learning impairments induced by scopolamine injection in mice

The authors studied the effects of T-588 on scopolamine-induced memory impairments in the acquisition of a classical eyeblink conditioning in behaving adult mice. Mice injected with 0.3 mg/kg of scopolamine showed a marked deficit, compared with nontreated mice, in the acquisition of classical eyeblink conditioning using a trace paradigm. Coadministration of T-588 (0.05% wt/vol, in water) with scopolamine (0.3 mg/kg) significantly prevented this deficit in associative learning. To further assess the effects of T-588 on motor coordination and the cognitive deficits induced by scopolamine, the authors compared adult controls or scopolamine-treated mice in different behavioral tasks: rotarod, object recognition, passive avoidance, and prepulse inhibition. In all of these tasks, the authors found a significant impairment in the motor or cognitive abilities in scopolamine-injected mice, compared with controls. In addition, the coadministration of T-588 with scopolamine restored deficits induced by scopolamine alone. Importantly, the administration of T-588 alone did not evoke any change compared with values obtained for controls. These results suggest that T-588 could be used as a pharmacological agent to improve motor and associative learning disorders.     

Use of mebendazole for helminthiases in chickens and geese

103 chickens experimentally infected with Capillaria obsignata were treated with Mebendazol at a dose of 30 mg/kg body-weight daily for 3 consecutive days. The effectiveness of the drug on the 4th stage larvae as well as immature and mature 5th stage forms were 92,5%, 98% and 98,8% respectively. In another experiment to 32 chickens experimentally infected with Syngamus trachea Mebendazol was given at 40 mg/kg body-weight on 3 successive days. Mebendazol removed the immature and mature gape worms completely. The anthelmintic effect of Mebendazol was also tested at 18 geese naturally infected with Amidostomum anseris, Trichostrongylus tenuis, Capillaria anatis, Hymenolepis lanceolata and Hymenolepis setigera. The treatment removed nematode-infection completely at a dose of 10 mg/kg body-weight and cestode-infection at a dose of 30 mg/kg body-weight when given daily for 3 consecutive days. Two groups of geese, 20 in each group, were treated against Hymenolepis-infection using Mebendazol in food for 6 consecutive days. It was found that Mebendazol when given at a dose of 10 mg/kg body-weight for 6 days removed the infection partly, whereas 30 mg/kg body-weight eliminated the whole worm burden.     

Cardioprotective activity of Ginkgo biloba Phytosomes in isoproterenol-induced myocardial necrosis in rats: A biochemical and histoarchitectural evaluation

The protective effects of Ginkgo biloba Phytosomes (GBP) in isoproterenol (ISO)-induced cardiotoxicity and the antioxidant activity involved in this protection were investigated in rats. Myocardial infarction was produced in rats with 65, 85, 120 and 200mg/kg of ISO administered subcutaneously (sc) twice at an interval of 24h. An ISO dose of 85mg/kg was selected for the present study as this dose offered significant alteration in biochemical parameters and moderate necrosis in heart. Effect of GBP oral treatment for 21 days at two doses (100mg and 200mg/kg body weight) was evaluated against ISO (85mg/kg, sc)-induced cardiac necrosis. Levels of marker enzymes (AST, LDH and CPK) were assessed in serum and heart, antioxidant parameters viz., reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) and malondialdehde (MDA) were assayed in heart homogenate. Significant myocardial necrosis, depletion of endogenous antioxidants and increase in serum levels of marker enzymes were observed in ISO-treated animals when compared with the normal animals. GBP elicited a significant cardioprotective activity by lowering the levels of serum marker enzymes and lipid peroxidation and elevated the levels of GSH, SOD, CAT, GPx and GR. The present findings have demonstrated that the cardioprotective effects of GBP in ISO-induced oxidative damage may be due to an augmentation of the endogenous antioxidants and inhibition of lipid peroxidation of membrane.     

Effects of PFOS and cyclophosphamide exposure on immune homeostasis in mice

Perfluorooctane sulfonic acid (PFOS) is member of a class of molecules with fluorinated carbon chains known as polyfluoroalkyls. PFOS have been used to produce a variety of industry and comsumer uses. However, a significant concern is that it accumulates in the environment, including in animals and humans, and that it is a potential immunosuppressant. Here we analyze immune homeostasis in mice following chronic exposure to PFOS at levels up to those historically found in PFOS manufacturing workers. Mice were exposed to 0.15, 1.5, 15, or 50 µg /kg of PFOS for 28 days, after which, B cells, T cells, and granulocytes from the bone marrow, liver, spleen, lymph nodes, and thymus were evaluated. We find that at these exposures, there was no effect of PFOS on major T- or B-cell populations, macrophages, dendritic cells, basophils, mast cells, eosinophils, neutrophils, serum antibodies or select serum cytokines. By contrast, mice exposed the known immunosuppressant cyclophosphamide, which was given at 40 mg/kg for four days, exhibited depletion of several granulocyte, T- and B-cell populations of the thymus, bone marrow, and spleen, as well as circulating IgM and IgE antibodies. These data indicate that exposures of up to 50 µg /kg of PFOS for 28 days does not affect immune homeostasis in mice.     

Effect of prolonged saline loading on HgCl2-induced renal tubular damage

Male Porton-Wistar rats, 32 weeks old, were given i.p. one of the following doses of HgCl2; 0.5, 1.0 or 1.5 mg Hg/kg. In the preceding 4-week period and throughout the experiment the animals had free access to either tap water or 1.0% saline. The urinary excretion of alkaline phosphatase measured in urine samples, collected during the first 24 h after treatment with mercury, indicated that chronic saline loading significantly attenuated tubular damage caused by 0.5 mg or 1.0 mg Hg/kg, but not by 1.5 mg Hg/kg. Tubular necrosis 12 and 24 h after mercury was also less severe and extensive in saline than in tap water-drinking rats. This difference was still noticeable 4 days after mercury treatment in rats dosed with 0.5 mg Hg/kg, but death in the two higher dose groups prevented further pair-to-pair histological comparison. At the selected dose levels chronic saline loading did not decrease renal mercury content at 12 or 24 h and therefore protection was not associated with decrease in renal mercury uptake. The experiment indicates that chronic saline drinking, which at higher doses attenuates HgCl2-induced acute renal failure but not tubular necrosis, is able to moderate the severity of tubular necrosis when the dose of HgCl2 is as low as 0.5 mg Hg/kg. This protective effect diminishes as the dose is increased.     

Induction of hepatic peroxisome proliferation in nonrodent species, including primates.

It is well established that the administration to rodents of a variety of structurally diverse chemicals possessing hypotriglyceridemic properties results in hepatomegaly, the induction of hepatic peroxisome (microbody) proliferation, and the development of hepatocellular carcinomas. Studies have led to the hypothesis that persistent proliferation of peroxisomes serves as an endogenous initiator of neoplastic transformation in liver by increasing the intracellular production of H2O2 by the peroxisomal oxidase(s). The objective of the present study was to determine whether hepatic peroxisome proliferation can be induced in cats, chickens, pigeons, and two species of monkeys (rhesus and cynomolgus). The hypolipidemic drug ciprofibrate (2-[4-(2,2-dichloro-cylopropyl)phenoxyl]2-methylpropionic acid) induced peroxisome proliferation in the livers of cats (dose, greater than 40 mg/kg body weight for 4 weeks); chickens (dose greater than 25 mg/kg body weight for 4 weeks); pigeons (300 mg/kg body weight for 3 weeks), rhesus monkeys (50 to 200 mg/kg body weight for 7 weeks) and cynomolgus monkeys (400 mg/kg body weight for 4 weeks). In all five species examined in this study, a marked but variable increase in the activities of peroxisomal catalase, carnitine acetyltransferase, heat-labile enoyl-CoA hydratase, and the fatty acid beta-oxidation system was observed. These results suggest that peroxisome proliferation can be induced in the livers of several species and that it is a dose-dependent but not a species-specific phenomenon.     

Effect of prolonged saline loading on HgCl2-induced renal tubular damage.

Male Porton-Wistar rats, 32 weeks old, were given i.p. one of the following doses of HgCl2; 0.5, 1.0 or 1.5 mg Hg/kg. In the preceding 4-week period and throughout the experiment the animals had free access to either tap water or 1.0% saline. The urinary excretion of alkaline phosphatase measured in urine samples, collected during the first 24 h after treatment with mercury, indicated that chronic saline loading significantly attenuated tubular damage caused by 0.5 mg or 1.0 mg Hg/kg, but not by 1.5 mg Hg/kg. Tubular necrosis 12 and 24 h after mercury was also less severe and extensive in saline than in tap water-drinking rats. This difference was still noticeable 4 days after mercury treatment in rats dosed with 0.5 mg Hg/kg, but death in the two higher dose groups prevented further pair-to-pair histological comparison. At the selected dose levels chronic saline loading did not decrease renal mercury content at 12 or 24 h and therefore protection was not associated with decrease in renal mercury uptake. The experiment indicates that chronic saline drinking, which at higher doses attenuates HgCl2-induced acute renal failure but not tubular necrosis, is able to moderate the severity of tubular necrosis when the dose of HgCl2 is as low as 0.5 mg Hg/kg. This protective effect diminishes as the dose is increased.