Protection by iron salts against sporidesmin intoxication in sheep

A number of iron compounds have been shown to protect sheep against the harmful effects of the facial eczema toxin, sporidesmin. Various salts were found to be effective; the oxidation state of the metal was not important although water-solubility did appear to be a prerequisite for prophylactic activity. The effect of iron salts was additive with that of zinc, and it is suggested that the protective action of these compounds results from their ability to inhibit the absorption of copper, consistent with the previously-proposed freeradical mechanism for sporidesmin toxicity.     

Urinary excretion of immunoreactive sporidesmin metabolites in sheep in relation to factors influencing susceptibility to sporidesmin intoxication

AIM: To study the urinary disposition of orally administered sporidesmins A and D in sheep and identify factors influencing their kinetics, particularly the influence of breeding for resistance and susceptibility to sporidesmin, the mycotoxin responsible for the hepatogenous photosensitisation, facial eczema. METHODS: A competitive ELISA was used to monitor urinary output of immunoreactive metabolites after the intraruminal administration, to female Romney sheep, of either sporidesmin A or sporidesmin D, the nontoxic analogue. Preliminary characterisation of metabolites was carried out using HPLC with fractions monitored by ELISA. RESULTS: Maximum urinary excretion rates of immunoreactive metabolites occurred 2-8 h after dosing with sporidesmin D and 15-30 h after dosing with sporidesmin A. Sporidesmin D caused no liver injury, as detected by changes in serum enzyme activity, while the liver injury caused by sporidesmin A was greatest for the sheep with the highest cumulative output of metabolite. When sporidesmin D was administered in two separate doses to sheep bred for either resistance or susceptibility to facial eczema, the variability of metabolic output between sheep within groups was much less after the second dose. The mean urinary metabolite excretion was greater for the susceptible than the resistant sheep but the difference was not significant. Potentiation (caused by pre-administration of small doses of sporidesmin A) resulted in a more severe reaction to the dosed sporidesmin A. Urinary output of metabolite was less in the potentiated than in the unpotentiated sheep. When resistant and susceptible sheep were dosed with sporidesmin A after potentiation there was no difference between them in their cumulative totals or excretion rates of immunoreactive metabolites. However, the volume of urine produced by the susceptible sheep was lower and less variable than the resistant sheep and consequently the concentration of their urinary metabolites was higher. Preliminary ELISA examination of HPLC-fractionated urine from a sheep dosed with sporidesmin A indicated the presence of several metabolites of sporidesmin. CONCLUSION: Sporidesmin A and metabolites are rapidly excreted in urine but not as rapidly as sporidesmin D and its metabolites. Only minor differences between sheep bred for resistance and susceptibility were seen. Potentiation caused a more severe reaction to sporidesmin A and less urinary excretion of the sporidesmin and its metabolites. CLINICAL RELEVANCE: This work is part of a programme with the aim of identifying FE-resistant animals without the need for sporidesmin dosing.     

An attempt to reproduce crystal-associated cholangitis in lambs in experimental dosing of sarsasapogenin or diosgenin alone and in combination with sporidesmin

No liver damage occurred in a group of 21 lambs dosed intraruminally with up to 9 g of sarsasapogenin or diosgenin daily for 10 consecutive days. In contrast, seven out of 15 lambs dosed with 0.1 mg of sporidesmin/kg liveweight in combination with sarsasapogenin and three out of six lambs dosed with sporidesmin in combination with diosgenin developed liver lesions. These were typical of those induced by sporidesmin. One lamb dosed with sporidesmin in combination with 9 g of diosgenin developed a crystal-associated cholangitis typical of Panicurn intoxication and alveld. No sapogenins were detected in urine by gas chromatography-mass spectrometry. The results suggest that orally administered sarsasapogenin and diosgenin are either not hepatotoxic per se or are too poorly absorbed to elicit a toxic response. The results provide only weak evidence that sporidesmin may be involved in the aetiology of Panicurn intoxication.     

Immunological studies of sporidesmin: Production of antibodies to azo-linked derivatives of 2-amino-5-chloro-3,4-dimethoxy benzyl alcohol

2-Amino-5-chloro-3,4-dimethoxy benzyl alcohol (ACD-MBA), a compound similar to part of the substituted tryptophan in the sporidesmin molecule was synthetized. It was coupled to bovine gamma-globulin through azo linkages and used to immunize guinea-pigs and rabbits or coupled to various bacteria and used to immunize guinea-pigs. Antibodies to ACDMBA were demonstrated by a complement fixation test. Hapten-inhibition of complement fixation showed that the anti-ACDMBA antibodies cross-reacted with sporidesmin and some derivatives of sporidesmin.     

Facial eczema in goats: the toxicity of sporidesmin in goats and its pathology

Groups of six goats were orally dosed with sporidesmin at rates of 0.3, 0.6, 1.2 and 2.4 mg of sporidesmin per kg body weight and their responses up to 6 weeks later compared with those of sheep dosed at the same time. Clinical facial eczema and pathological lesions similar to those found in sheep were found in all the goat breeds, but at higher dose rates of sporidesmin than those which caused equivalent lesions in sheep. Saanens were the most susceptible goat breed, requiring 2-4 times as much sporidesmin as sheep to achieve similar effects. G4 and feral goats required 4-8 times the sheep dose of sporidesmin to obtain similar responses. Gamma-glutamyltransferase reached its highest serum levels after 20 days while glutamate dehydrogenase and aspartate aminotransferase reached their highest levels between 10 and 20 days. Alkaline phosphatase did not rise consistently to high levels in affected goats. The elevation in aspartate aminotransferase levels tended to be early and transient; glutamate dehydrogenase early and prolonged; gamma-glutamyltransferase late and prolonged, and'alkaline phosphatase late and minor. There was considerable individual variation in the time at which elevations occurred and the levels which enzymes reached. Cholesterol and bilirubin levels were high if liver injury was severe.     

Effects of low dose rates of sporidesmin given orally to sheep

Aim: To examine clinical and subclinical effects of sporidesmin administered orally to sheep at very low daily dose rates for periods of 3 to 48 days.

Methods: Two experiments were conducted. In Experiment A, sporidesmin-A was administered orally to groups of 16 sheep at daily dose rates of approximately 0.0042, 0.0083 and 0.0167 mg/kg bodyweight for 48 days. In Experiment B, the highest of these doses was administered orally for 3, 6, 12, 24 or 48 consecutive days. Parameters of production, clinical findings, organ weights and pathological findings were recorded.

Results: In Experiment A, severe liver lesions and photosensitisation were evident as early as 18 days after commencement of daily low-dose administration of sporidesmin, and were associated with significant bodyweight loss. Significant bodyweight loss also occurred in non-photosensitised sporidesmin-treated sheep. Bodyweight reductions were associated with reduced carcass weights and skin weights in treated animals. Sporidesmin administration was also associated with reduced bodyweight gains and pathological changes of the liver, kidney, hepatic lymph nodes, thymus, adrenal gland, heart and spleen. In Experiment B, only moderate changes occurred in a few sheep in the groups dosed with sporidesmin at 0.0167 mg/kg for 3 or 6 days, but major changes were frequently recorded in animals dosed at this rate for 12 days or longer. These comprised changes in the liver and other organs, and photosensitisation typical of the disease, facial eczema. Results are discussed in relation to animal welfare and economic issues associated with this disease.

Conclusions: Sporidesmin caused significant clinical and sub-clinical disease and reduced animal production at relatively low daily dose rates. The effects of repeated daily low-dose administration of sporidesmin appear to be cumulative. There was considerable variation in susceptibility between individual animals.These results emphasise the considerable production losses and animal welfare effects associated with sporidesmin toxicity in sheep.     

The protective effect of zinc sulphate in experimental sporidesmin poisoning of sheep

Aqueous solutions of zinc sulphate were administered orally to sheep over 5 days (0.125, 0.5 and 2.0 g Zn⁺⁺/sheep/day) to bracket a 3-day period during which sporidesmin also was dosed. The zinc sulphate treatment gave protection from the effects of sporidesmin when compared with control groups dosed sporidesmin alone. Body weight changes were improved and liverdamage scores, numbers of animals showing photosensitisation, serum levels of glutamic oxaloacetic acid transaminase and total bilirubin were lower. The protective effects of zinc sulphate were obtained at levels well abovethose required for growth and maintenance. Protection increased but at a diminishing rate with increasing dose rate of zinc. Because of the small safety margin which exists between the dose rate of zinc sulphate which will provide adequate protection and that which will cause toxicity the use of zinc for the control of facial eczema is not recommended.     

A review of developments in nutrition as it is related to fertility in cattle: 1964–9

Mice given sporidesmin in their drinking water for 10 days developed bilirubinaemia. Mice immunized with some 2-amino-5-chloro-3, 4-dimethoxy benzyl alcohol-bacteria complexes were apparently more resistant to the bilirubinaemic effect of sporidesmin than were unimmunized mice.     

Uniformity of response of identical twin cattle to sporidesmin intoxication

Sixteen pairs of identical twin dairy calves were reared together and at one year of age subjected to a test of uniformity of response to sporidesmin intoxication. The response was measured by scoring livers for toxic injury and by measuring serum gamma-glutamyltransferase at weekly intervals after dosing. Within pair variance in serum gamma-glutamyltransferase was greatest at two and six weeks after sporidesmin dosing when enzyme levels were rising and falling. Within twin variance and the interclass correlation coefficients were least and greatest respectively at five weeks after dosing. At five weeks the GGT correlation coefficient was 0.89 and for liver injury score 0.76. High consistency among twins of a pair, compared with differences among pairs indicates a high relative efficiency in using identical twins rather than singles for experimental work (approximately x 5). It is also consistent with a high heritability of resistance to sporidesmin intoxication in dairy cattle.     

Effects of high iron and sulfate ion concentrations on dry matter digestion and volatile fatty acid production by ruminal microorganisms.

The inhibitory effects of iron- and sulfate-containing compounds on the in vitro digestion of a balanced forage diet by mixed populations of ruminal microorganisms were examined in batch cultures. Compounds containing ferrous and ferric cations consistently inhibited DM digestion by up to 36% when added Fe concentrations in cultures were between 100 and 1,000 mg/L. Increased sulfate concentrations of up to 200 mg/L or chloride concentrations of up to 635 mg/L were not associated with decreased DM digestion. Ammonium sulfate additions that provided 200 mg/L of added sulfur increased (P less than .05) digestibility by 10%. Sulfate-containing iron salts tended to be less inhibitory than chloride salts and were associated with increased gas production during digestion. Ferric chloride inhibited (P less than .05) microbial activities at lower concentrations than ferrous chloride. Data suggest that excessive iron supplementation or contamination of feeds with iron-containing pollutants may decrease microbial activities in the rumen.     

Putative sporidesmin toxicity in an Eastern Grey kangaroo (Macropus giganteus)

A 2-year-old, captive, male Eastern Grey kangaroo (Macropus giganteus) died after progressive weight loss over a 4 week period. Biochemical analysis suggested hepatobiliary injury. At necropsy the liver was small, pale and firm. There were no abnormalities detected in other organs. Histopathological examination revealed a severe, diffuse, obliterative cholangiohepatopathy with advanced periportal fibrosis. This chronic hepatotoxicity was consistent with exposure to sporidesmin, the toxic metabolite in the spores of the fungus Pithomyces chartarum. Restricted grazing opportunities and heavy fungal pasture contamination may have precipitated sporidesmin toxicity in this animal. Sporidesmin toxicity has not previously been reported in this species.     

Effects of low dose rates of sporidesmin given orally to sheep

AIM: To examine clinical and subclinical effects of sporidesmin administered orally to sheep at very low daily dose rates for periods of 3 to 48 days. METHODS: Two experiments were conducted. In Experiment A, sporidesmin-A was administered orally to groups of 16 sheep at daily dose rates of approximately 0.0042, 0.0083 and 0.0167 mg/kg bodyweight for 48 days. In Experiment B, the highest of these doses was administered orally for 3, 6, 12, 24 or 48 consecutive days. Parameters of production, clinical findings, organ weights and pathological findings were recorded. RESULTS: In Experiment A, severe liver lesions and photosensitisation were evident as early as 18 days after commencement of daily low-dose administration of sporidesmin, and were associated with significant bodyweight loss. Significant bodyweight loss also occurred in non-photosensitised sporidesmin-treated sheep. Bodyweight reductions were associated with reduced carcass weights and skin weights in treated animals. Sporidesmin administration was also associated with reduced bodyweight gains and pathological changes of the liver, kidney, hepatic lymph nodes, thymus, adrenal gland, heart and spleen. In Experiment B, only moderate changes occurred in a few sheep in the groups dosed with sporidesmin at 0.0167 mg/kg for 3 or 6 days, but major changes were frequently recorded in animals dosed at this rate for 12 days or longer. These comprised changes in the liver and other organs, and photosensitisation typical of the disease, facial eczema. Results are discussed in relation to animal welfare and economic issues associated with this disease. CONCLUSIONS: Sporidesmin caused significant clinical and sub-clinical disease and reduced animal production at relatively low daily dose rates. The effects of repeated daily low-dose administration of sporidesmin appear to be cumulative. There was considerable variation in susceptibility between individual animals. These results emphasise the considerable production losses and animal welfare effects associated with sporidesmin toxicity in sheep.     

The protective effect of zinc sulphate in experimental sporidesmin intoxication of lactating dairy cows

Zinc sulphate solution, administered concurrently with the mycotoxin sporidesmin, gave significant protection against the toxin. The protective effect was shown in maintained milk production and bodyweights, and in reduced liver damage as determined by serum enzyme (gamma-glutamyltransferase, ornithine carbamyltransferase) analysis and by subjective grading of the liver damage after slaughter. There was no overt facial eczema in either group but, in sporidesmin dosed cows not receiving zinc sulphate, there was a fall in milk yield and in bodyweight. Serum enzyme levels did not rise until more than a week after dosing at which time milk yields were showing partial recovery. Serum concentrations of the enzyme gamma-gluta-myltransferase (EC 23.2.2) were found to be correlated to the severity of the liver damage observed at post-mortem.     

Lack of toxicity of a nonsporidesmin-producing strain of Pithomyces chartarum in cell culture and when dosed to lambs

In New Zealand the fungus Pithomyces charturum normally produces sporidesmin, a mycotoxin, which is responsible for the hepatogenous photosensitisation disease known as facial eczema. Cultures from an isolate of P. charturum, which does not produce sporidesmin, were examined by cell culture and by dosing to lambs to determine whether other toxic metabolites were produced. Acute and long term toxicity studies were conducted with the toxic response being assessed by weight changes, postmortem and histological examination of tissues, blood biochemistry and haematology tests.

An extract from a sporidesmin-producing isolate was highly toxic in cell culture, while extracts of the nonsporidesmin-producing isolate did not cause a cytotoxic response to HEp 2 cells.

After dosing with a sporidesmin-producing isolate, lambs developed liver lesions and clinical signs of facial eczema. Serum biochemistry changes occurred which were consistent with sporidesmin poisoning.

Lambs dosed with the nonsporidesmin-producing isolate, at the rate of thirty times the number of spores of the sporidesmin-producing isolate, showed no observable toxic effects. All organs were of normal appearance, and histological examination of tissues, blood biochemistry and haematology results showed no abnormal changes. Similarly, long term dosing of extracts of the nonsporidesmin-producing isolate, at a rate equivalent to 100 000 spores/g of grass, produced no indication of a toxic response. It was concluded that the nonsporidesmin-producing isolate of P. churtarum contained no toxic metabolites in significant concentration.     

Lack of toxicity of a non-sporidesmin-producing strain of Pithomyces chartarum in cell culture and when dosed to lambs

In New Zealand the fungus Pithomyces charturum normally produces sporidesmin, a mycotoxin, which is responsible for the hepatogenous photosensitisation disease known as facial eczema. Cultures from an isolate of P. charturum, which does not produce sporidesmin, were examined by cell culture and by dosing to lambs to determine whether other toxic metabolites were produced. Acute and long term toxicity studies were conducted with the toxic response being assessed by weight changes, postmortem and histological examination of tissues, blood biochemistry and haematology tests. An extract from a sporidesmin-producing isolate was highly toxic in cell culture, while extracts of the nonsporidesmin-producing isolate did not cause a cytotoxic response to HEp 2 cells. After dosing with a sporidesmin-producing isolate, lambs developed liver lesions and clinical signs of facial eczema. Serum biochemistry changes occurred which were consistent with sporidesmin poisoning. Lambs dosed with the nonsporidesmin-producing isolate, at the rate of thirty times the number of spores of the sporidesmin-producing isolate, showed no observable toxic effects. All organs were of normal appearance, and histological examination of tissues, blood biochemistry and haematology results showed no abnormal changes. Similarly, long term dosing of extracts of the nonsporidesmin-producing isolate, at a rate equivalent to 100,000 spores/g of grass, produced no indication of a toxic response. It was concluded that the nonsporidesmin-producing isolate of P. churtarum contained no toxic metabolites in significant concentration.     

The cellular and molecular toxicity of sporidesmin

ABSTRACT

The fungal metabolite sporidesmin is responsible for the hepatogenous photosensitising disease facial eczema in livestock. Toxicity is due to a sulfur-bridged epidithiodioxopiperazine ring that has wide biological reactivity. The ways in which the toxin causes hepatobiliary and other tissue damage have not been established. Hypotheses include direct interaction with cellular thiols including protein cysteine residues or production of reactive oxygen species resulting in oxidative stress. Comparison with the cellular effects of the structurally related compound gliotoxin suggests additional mechanisms including interaction with cell adhesion complexes and possible downstream consequences for regulated necrosis as a response to tissue injury. Revision of hypotheses of how sporidesmin affects cells has the potential to generate new strategies for control of facial eczema including through identification of proteins and genes that are associated with resistance to the disease.     

Serum gamma-glutamyltransferase as a measure of sporidesmin-induced liver damage in sheep

The relationship between serum concentrations of the enzyme gamma-glutamyltransferase (GGT) after experimental sporidesmin intoxication, and the severity of the liver damage seen on post-mortem examination of the liver, has been examined. Serum GGT activity in blood collected 2 to 3 weeks after ad-ministering the toxin is positively correlated to a subjective liver damage score and to losses in bodyweight associated with the intoxication. Determination of GGT activities provides a sensitive method for detecting and measuring liver damage in experimental sporidesmin poisoning. In the field, GGT determinations should prove useful in diagnosing liver damage associated with subclinical and clinical facial eczema giving additional information about the severity of the lesion.     

A zinc-containing intraruminal device for prevention of the sporidesmin-induced cholangiopathy of facial eczema in calves

AIM: To develop and evaluate a zinc-containing intraruminal controlled-release bolus for protection of calves (175-250 kg bodyweight) against facial eczema (FE). METHODS: Boluses releasing zinc, in the form of zinc oxide, at rates ranging from 1.67 to 4.25 g/day were administered to calves which were challenged 4 weeks later with the FE toxin, sporidesmin. The efficacy of the boluses in protecting against sporidesmin-induced cholangiopathy was determined by measuring serum activities of gamma-glutamyltransferase (GGT). RESULTS: A bolus releasing zinc at approximately 4.25 g/day gave excellent protection against sporidesmin toxicity for periods of up to 5 weeks duration. CONCLUSIONS: This zinc-containing intraruminal controlled-release bolus has the potential to markedly reduce the incidence and severity of FE in calves within a 175-250 kg bodyweight range.     

Glycogen accumulation and histological changes in the livers of lambs with alveld and experimental sporidesmin intoxication

Alveld is a hepatogenous photosensitization disease seen in lambs grazing Narthecium ossifragum pastures in Norway. Mycotoxins, possibly sporidesmin, have been suspected to cause the liver damage in alveld as in facial eczema. The histological changes in the liver of alveld cases and in lambs photosensitized after experimental sporidesmin intoxication were compared. The liver damage characterized by necrosis in single centrilobular hepatocytes, was of the same type in both conditions. Minor to moderate portal fibroplasia and bile duct proliferation were almost always present. Accumulated glycogen was seen in hepatocytes in the centrilobular areas. This was significantly correlated to the enzymatically measured glycogen content and there was good correlation between parenchymal damage and glycogen accumulation. The glucose-6-phosphatase and glycogen phosphorylase activities were normal. These findings indicate that parenchymal damage, rather than obstruction of the bile ducts, caused the retention of phylloerythrin both in alveld cases and in experimentally sporidesmin-intoxicated lambs. The accumulation of glycogen could not be explained.Abbreviations HE haematoxylin and eoson - PAS periodic acid Schiff - EDTA ethylene diamine tetracetic acid     

Inheritance of resistance to facial eczema: a review of research findings from sheep and cattle in New Zealand

Facial eczema (FE) is a costly problem to New Zealand pastoral agriculture, and has a detrimental impact on animal wellbeing. Incidence and severity of the disease can be reduced by grazing management and zinc prophylaxis. An additional strategy is to breed animals that are genetically resistant to intoxication with sporidesmin, the causative mycotoxin. This review summarises research findings on the inheritance of resistance of animals to FE, including evidence of among- and within-breed genetic variation, direct and correlated responses to selection, and identification of genetic markers and candidate genes for FE resistance.