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.     

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     

Competition of a sporidesmin-producing Pithomyces strain with a non-toxigenic Pithomyces strain

Sporidesmin, a mycotoxin produced by some strains of Pithomyces chartarum, is responsible for the hepatogenous photosensitisation disease facial eczema, which causes severe losses in agricultural revenue in New Zealand. A sporidesmin-producing strain of P. chartarum, isolated in New Zealand, was grown in co-culture with a South African strain that does not produce the mycotoxin. Competition occurred between the two strains when grown both on agar plates and on dried ryegrass, with a significant decrease in the total amount of sporidesmin produced. Biological control of toxkenic P. chartarum can thus occur under laboratory conditions, raising the possibility of similar control in the field situation.     

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.     

The occurrence of Anthrax in New Zealand

A field outbreak of facial eczema occurred during a vitamin B12 response trial in young growing sheep. Pasture cobalt levels were in the low range for sheep <0.08 mg/kg, 1.358 µmol/kg) and mean (of 3) liver vitamin B12 levels in the sheep were low (>100 nmol/kg) during the period in which facial eczema occurred.

Mean serum vitamin B12 levels of the untreated group were low (>185 pmol/1) for the two months (January and February) preceding the period of facial eczema. However, levels showed an approximate 3.5 fold increase in both cobalt supplemented and unsupplemented groups with the onset of facial eczema in March. From February to March the mean serum vitamin B12 and glutamyl transferase (GGT) activity showed parallel increases with a positive correlation (r = 0.73) between log serum vitamin B12 and log serum GGT activity during the period January to July for both groups. This finding suggested that the increase in serum vitamin B12 was due to sporidesmin induced liver damage.

The diagnostic implication is that, in areas where facial eczema is a problem, liver is the sample of choice for determining vitamin B12 status, because sporidesmin toxicity can elevate low serum vitamin B12 levels to diagnostically normal levels.     

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.     

Competition of a sporidesmin-producing Pithomyces strain with a non-toxigenic Pithomyces strain

Sporidesmin, a mycotoxin produced by some strains of Pithomyces chartarum, is responsible for the hepatogenous photosensitisation disease facial eczema, which causes severe losses in agricultural revenue in New Zealand. A sporidesmin-producing strain of P. chartarum, isolated in New Zealand, was grown in co-culture with a South African strain that does not produce the mycotoxin. Competition occurred between the two strains when grown both on agar plates and on dried ryegrass, with a significant decrease in the total amount of sporidesmin produced. Biological control of toxigenic P. chartarum can thus occur under laboratory conditions, raising the possibility of similar control in the field situation.     

Spectrofluorometric analysis of phylloerythrin (phytoporphyrin) in plasma and tissues from sheep suffering from facial eczema

AIMS: To study the increase in phylloerythrin concentration in plasma and the disposition of phylloerythrin in skin and other tissues of sheep in which the hepatogenous photosensitisation,facial eczema, had been experimentally induced by dosing with the mycotoxin, sporidesmin. Spectroscopic differences between plasma and skin measurements of animals kept inside and outside after dosing were also studied in order to establish whether phylloerythrin undergoes photodegradation when exposed to sunlight. METHODS: Twenty-six Romney x Polled Dorset (25-30 kg)weaned female lambs were purchased from a commercial flock in the Waikato region, New Zealand. Twenty-two of these lambs were dosed with 0.25 mg sporidesmin/kg liveweight on each of two consecutive days (Days -1 and 0); the remaining four lambs served as controls. Both sporidesmin-dosed lambs and controls were randomly divided into two penned groups, one group housed inside in a darkened room and the others outside, exposed to natural sunlight. The lambs were fed green lucerne pellets and lucerne chaff ad libitum for 10 days prior to dosing and until Day 12 after the first dose; thereafter, all the lambs were fed fresh, cut grass (mainly ryegrass) ad libitum, until the end of the experimental period on Day 26. Plasma samples collected on Days -2, 7, 10, 12, 14, 17, 20 and 25were analysed for gamma glutamyltransferase (GGT) activity, bilirubin concentration, and the fluorescence spectrum of phylloerythrin. Spectrofluorometric analysis of phylloerythrin in skin was performed in vivo on the same days, using an external fiber-optic probe. RESULTS: Eight of 11 lambs (73%) kept outside after sporidesmin dosing became photosensitised during the experimental period. None of the sporidesmin-dosed animals kept inside showed clinical signs of photosensitisation. The GGT activity in plasma increased exponentially during the experimental period in all sporidesmin-dosed animals until it reached a plateau. All plasma obtained from sporidesmin-dosed sheep had spectral characteristics similar to those of phylloerythrin, namely a peak in the excitation spectrum at 422 nm and strong emission band at 650 (SE 1) and 709 (SE 1) nm. The fluorescence under excitation at 422 nm of phylloerythrin added to plasma from control lambs had identical peaks. Emission spectra obtained from plasma from healthy sheep without addition of phylloerythrin showed either no fluorescence or minor fluorescence at around 671 nm. Fluorescence in skin of sporidesmin-dosed animals had similar spectra to that in plasma. The appearance of the phylloerythrin-like spectra occurred 2-3 days later in the skin than in plasma, and phylloerythrin in sunlight-exposed skin did not suffer photodegradation during the course of the study. CONCLUSION: Plasma concentrations of phylloerythrin in healthy sheep were <0.1 micromol/l, and clinical signs of photosensitisation were not evident until concentrations exceeded 0.3 micromol/l. Plasma concentrations of phylloerythrin rose as high as 4.9 micromol/l in some animals. The concentration of phylloerythrin in skin began increasing 2-3 days later than that in blood. Hepatogenous photosensitisation can be diagnosed by analysis of plasma phylloerythrin concentrations using a spectroscopic method.     

The effect of sporidesmin toxicity on ovine serum vitamin B12 levels

A field outbreak of facial eczema occurred during a vitamin B12 response trial in young growing sheep. Pasture cobalt levels were in the low range for sheep (<0.08 mg/kg, 1.358 micromol/kg) and mean (of 3) liver vitamin B12 levels in the sheep were low (<400 nmol/kg) during the period in which facial eczema occurred. Mean serum vitamin B12 levels of the untreated group were low (<485 pmol/l) for the two months (January and February) preceding the period of facial eczema. However, levels showed an approximate 3.5 fold increase in both cobalt supplemented and unsupplemented groups with the onset of facial eczema in March. From February to March the mean serum vitamin B12 and glutamyl transferase (GGT) activity showed parallel increases with a positive correlation (r = 0.73) between log serum vitamin B12 and log serum GGT activity during the period January to July for both groups. This finding suggested that the increase in serum vitamin B12 was due to sporidesmin induced liver damage. The diagnostic implication is that, in areas where facial eczema is a problem, liver is the sample of choice for determining vitamin B12 status. because sporidesmin toxicity can elevate low serum vitamin B12 levels to diagnostically normal levels.     

First case of pithomycotoxicosis (facial eczema) in the Netherlands

Between mid September and the beginning of November 2005, the Animal Health Service (AHS) received thirteen reports offarms on which several animals showed severe symptoms of solar eczema. Blood chemistry showed very high levels of GOT/AST and GGT indicative of severe liver damage. Farm visits to eight farms showed that the animals--previous to the start of the symptoms--had been grazing 24 hours/day and received no additional feed. Ingestion of poisonous plants or medications was considered unlikely to have caused the liver damage, and liver fluke infections were present on only two farms. Microscopic examination of specimens of grass revealed the presence of spores of Pithomyces chartarum in samples taken from six of nine farms. This fungus produces the mycotoxin sporidesmin, which causes severe liver damage and pithomycotoxicosis (facial eczema). This article is the first to describe Pithomyces chartarum in cattle in mainland Europe. Further research on the distribution and re-occurrence of Pithomyces chartarum infection and sporidesmin survival in grass silage is recommended.     

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.     

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.     

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.     

Excretion of loline alkaloids in urine and faeces of sheep dosed with meadow fescue (Festuca pratensis) seed containing high concentrations of loline alkaloids

Abstract

AIM: To determine the effect of oral dosing of sheep with loline alkaloids on their excretion in urine and faeces, and to monitor for any toxic effects.

METHODS: In Experiment 1, six 9-month-old ewe lambs were given a single oral dose of loline alkaloids (52 mg/kg bodyweight (BW); acute exposure) as a suspension of ground meadow fescue (Festuca pratensis) seed in water. In Experiment 2, on six consecutive days, six ewe lambs were given three doses of loline (68 mg/kg BW/day; chronic exposure). Blood was collected at variable intervals up to 72 h in Experiment 1, and up to 8 days in Experiment 2, for haematology and measurement of alkaline phosphatase, aspartate aminotransaminase, creatine kinase and γ-glutamyl transferase in plasma. Urine and faecal samples were collected at similar times for measurement of creatinine in urine and loline alkaloid analysis. A post mortem with histopathology was carried out on two animals at the end of each experiment.

RESULTS: The loline alkaloids, N-acetyl norloline, N-formyl loline, N-acetyl loline, N-methyl loline and loline base were detected in urine within 15 minutes after the single dosing. N-formyl loline and loline base were the predominant metabolites in urine in both experiments. The total quantity of lolines excreted in both urine and faeces was 10% and 4% of the amount dosed in Experiments 1 and 2, respectively. In both experiments, the clinical chemistry parameters in blood and urine were within normal ranges. Post-mortem and histopathological examination did not show any abnormalities.

CONCLUSIONS: This is the first report of loline alkaloid profiles in both urine and faeces of sheep. The appearance of loline alkaloids and the loline base in urine within 15 minutes suggests rapid uptake, metabolism and excretion. Loline alkaloids were non-toxic to sheep at the concentrations they are exposed to under New Zealand grazing conditions. The low recovery of loline alkaloids in urine and faeces in the absence of toxicity signs suggests lolines are extensively metabolised; probably to forms other than N-formyl loline, N-methyl loline, N-acetyl loline, N-acetyl norloline, and loline base in the digestive tract of sheep prior to absorption, and/or in the liver or other tissues following absorption.     

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 influence of lime and nitrogen fertilisers on spore counts of Pithomyces chartarum in pasture

AIMS: To determine whether the application of lime or nitrogen to pasture affected the spore counts of Pithomyces chartarum.

MATERIALS AND METHODS: The lime application studies were undertaken on a spring-calving, pasture-based, commercial dairy farm near Te Awamutu, New Zealand. On 6 November 2012, five randomly selected paddocks were split into three equal sections. In two of the sections, lime was applied at either 1.5 or 2.5?t/ha, and the central section was left as an untreated control. Each section was sampled for spore counting weekly from 16 January to 15 May 2013.

Starting in January 2013, five other randomly selected paddocks were monitored for spore counts. On 20 March 2013 the average spore counts in three paddocks were >100,000 spores/g of pasture. These paddocks were then divided into three equal sections and lime was applied as described above. Spore counting in each section continued weekly until 15 May 2013.

The nitrogen application study was carried out on three commercial dairy farms near Te Awamutu, New Zealand. Two randomly selected paddocks on each farm were divided into three equal sections and, on 20 December 2012, nitrogen in the form of urea was applied at either 50 or 80?kg urea/ha to two of the sections; the central section remained as an untreated control. Each section was sampled for spore counting weekly from 16 January to 15 May 2013.

RESULTS: Following pre-summer lime application, treatment at 1.5 or 2.5?t/ha did not affect spore counts over time compared with the control section (p>0.26). Similarly following autumn lime application, treatment at 1.5 or 2.5?t/ha did not affect spore counts over time compared with the control section (p>0.11). Following nitrogen application median spore counts remained <20,000 spores/g pasture throughout the trial period and there was no effect of treatment on spore counts over time (p>0.49).

CONCLUSION: This study found that application of lime before the risk period for facial eczema, in November, application of lime after a spore count rise, in March, or urea application in December did not affect changes in number of spores produced by P. chartarum.

CLINICAL RELEVANCE: This study does not support previous suggestions that fertilising pasture with lime or urea could alter the spore counts of P. chartarum. Fertiliser use does not provide an alternative to, or support, conventional methods of facial eczema control such as zinc prophylaxis or treatment of pasture with fungicides.     

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.     

Pithomycotoxicosis (facial eczema) in ruminants in the Azores, Portugal

Outbreaks of pithomycotoxicosis (facial eczema), a hepatogenous photosensitisation caused by the mycotoxin sporidesmin, have affected ruminants in the Azores Islands of Portugal after warm, humid periods during late summer and autumn. Twenty-two outbreaks were recorded in cattle between 1999 and 2001, affecting 11.4 per cent of the animals in the affected herds, and in 2000 there was an outbreak in one sheep flock in which more than 20 per cent of the sheep died. The clinical signs included decreases in milk production, weight loss, photosensitisation and its sequelae, including death. The animals had high activities of gamma glutamyltransferase in their serum, and icterus and severe liver disease, including biliary hyperplasia and fibrosis, were found postmortem. The characteristic spores of the toxigenic saprophytic fungus Pithomyces chartarum were found on grass; all 381 isolates of the fungus were toxigenic for sporidesmin by elisa, and the results were confirmed by high-performance liquid chromatography analysis. Cattle from farms at greatest risk of pithomycotoxicosis were protected by supplementing their concentrate feed with zinc oxide, or using a slow-release intraruminal zinc bolus.     

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.     

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 free-radical mechanism for sporidesmin toxicity.