Chronic pithomycotoxicosis associated with obstructive rhinopathy in sheep.

Pithomycotoxicosis (facial eczema) is a seasonal hepatogenous photosensitization of sheep caused by the ingestion of sporidesmin contained in the spores of the fungus Pithomyces chartarum. We describe 4 cases of obstructive rhinopathy associated with chronic pithomycotoxicosis naturally occurring in the north of Spain. Sheep were 5 to 7 years old and Latxa breed. A detailed clinical study was conducted together with computerized tomography examination and completed by necropsy and histopathology. All sheep developed a permanent narrowing of the nasal lumen close to the nostrils causing inspiratory dyspnea and snoring. Computerized tomography demonstrated a significant increase of soft tissue in the rostral nasal cavity. Elevated gamma-glutamyl transferase, alanine aminotransferase, and lipase were noted on serum biochemistry. At necropsy, liver atrophy and fibrosis associated with chronic pithomycotoxicosis was identified in 3 of the sheep. All sheep had whitish elevations and rough surfaces on the alar folds and areas adjacent to the nasal surfaces. Histopathologic assessments, which included histochemical and immunohistochemical techniques, of the nasal lesions identified moderate to severe arteriosclerosis in 21.5% to 61.9% of the small arteries evaluated with surrounding fibrosis and edema. No changes associated with hypersensitivity reactions were found. These lesions were similar to the ones described in blood vessels of the liver in chronic pithomycotoxicosis and in our cases. The results of this study suggest a direct action of the sporidesmin on the rostral nasal cavity. Further studies are needed to analyze the impact of the sporidesmin on the sheep nasal mucosa.

Longitudinal study of the effect of sporidesmin toxicity on lamb production and serum biochemistry in a flock of 46 Romney ewes using a standardised measure of liver damage.

AIMS: To evaluate the effect of sporidesmin toxicity on production outcomes and serum biochemistry analytes in mixed age Romney ewes, using a standardised measure of liver damage. METHODS: This was a prospective longitudinal study following 46 mixed age Romney ewes from sporidesmin intoxication in April 2019, to slaughter 8 months later. The ewes were blood-sampled up to eight times, with a panel of serum biochemistry tests performed on the final six samples. However, only gamma-glutamyl transferase (GGT) activity was measured in the first two samples collected at the end of sporidesmin intoxication and 2 weeks later. Body condition score, ewe weight and production data were also recorded. Using a standardised liver score, based on histology of liver samples collected at slaughter, ewes were assigned to one of three liver disease categories (LDC); low, middle, and high. These were then used as the outcome or predictor variables for statistical analyses. Finally, two separate decision tree models, using recursive partitioning (RP), were fitted to the biochemistry data and to the GGT data collected at FE outbreak, to predict ewes in the low LDC. RESULTS: There was no evidence of a difference for the effect of LDC on ewe weight (p = 0.86) with ewes, on average, gaining weight to weaning. Weaning percent, lamb rearing percent and ewe flock efficiency were lower in ewes with high LDC, and scanning-to-weaning lamb loss was significantly higher in sheep with high LDC (p = 0.02). Serum activities of GGT and glutamate dehydrogenase and concentration of globulin were significantly lower in sheep with low LDC than in sheep with middle or high LDC (p < 0.05). However, there was no evidence of a difference for the effect of LDC on other biochemistry variables (p > 0.05). The final RP model for the biochemistry data categorised ewes as low LDC if their GGT was <122 IU/L, 3 months after sporidesmin intoxication, or if their GGT was <514 IU/L, <18 days after sporidesmin intoxication. CONCLUSIONS AND CLINICAL RELEVANCE: Sheep with gross and histological evidence of severe sporidesmin-induced liver damage were able to maintain or gain body weight, suggesting that sporidesmin intoxication alone is not causative of poor body condition. Similarly, many of the serum biochemistry tests were not associated with evidence of liver damage. Lamb production was reduced in ewes with evidence of severe liver damage and the decision tree model showed promise as a basis to select ewes for culling.

Genomic Tools for the Identification of Loci Associated with Facial Eczema in New Zealand Sheep.

Facial eczema (FE) is a significant metabolic disease that affects New Zealand ruminants. Ingestion of the mycotoxin sporidesmin leads to liver and bile duct damage, which can result in photosensitisation, reduced productivity and death. Strategies used to manage the incidence and severity of the disease include breeding. In sheep, there is considerable genetic variation in the response to FE. A commercial testing program is available for ram breeders who aim to increase tolerance, determined by the concentration of the serum enzyme, gamma-glutamyltransferase 21 days after a measured sporidesmin challenge (GGT21). Genome-wide association studies were carried out to determine regions of the genome associated with GGT21. Two regions on chromosomes 15 and 24 are reported, which explain 5% and 1% of the phenotypic variance in the response to FE, respectively. The region on chromosome 15 contains the ß-globin locus. Of the significant SNPs in the region, one is a missense variant within the haemoglobin subunit ß (HBB) gene. Mass spectrometry of haemoglobin from animals with differing genotypes at this locus indicated that genotypes are associated with different forms of adult ß-globin. Haemoglobin haplotypes have previously been associated with variation in several health-related traits in sheep and warrant further investigation regarding their role in tolerance to FE in sheep. We show a strategic approach to the identification of regions of importance for commercial breeding programs with a combination of discovery, statistical and biological validation. This study highlights the power of using increased density genotyping for the identification of influential genomic regions, combined with subsequent inclusion on lower density genotyping platforms.

Primary Impacts of the Fungal Toxin Sporidesmin on HepG2 Cells: Altered Cell Adhesion without Oxidative Stress or Cell Death.

The fungal metabolite sporidesmin is responsible for severe necrotizing inflammation of biliary tract and liver of livestock grazing on pasture containing spores of Pithomyces chartarum that synthesizes the toxin. The toxin is secreted into bile causing the erosion of the biliary epithelium accompanied by inflammation and damage to surrounding tissues. Toxicity has been suggested to be due to cycles of reduction and oxidation of sporidesmin leading to oxidative damage from the formation of reactive oxygen species. The current work is the first test of the oxidative stress hypothesis using cultured cells. Oxidative stress could not be detected in HepG2 cells incubated with sporidesmin using a dichlorodihydrofluorescein diacetate assay or by use of two-dimensional electrophoresis to search for oxidized peroxiredoxins. There was also no evidence for necrosis or apoptosis, although there was a loss of cell adhesion that was accompanied by the disruption of intracellular actin microfilaments that have known roles in cell adhesion. The results are consistent with a model in which altered contact between cells in situ leads to altered permeability and subsequent inflammation and necrosis, potentially from the leakage of toxic bile into surrounding tissues. There is now a need for the further characterization of the damage processes in vivo, including the investigation of altered permeability and mechanisms of cell death in the biliary tract and other affected organs.

Chronic facial eczema in sheep: description of gross and histological changes in the liver and association with serum gamma-glutamyltransferase activity at the time of sporidesmin intoxication.

AIMS: To determine the gross and histological changes developing in the liver of sheep 8 months after a single period of exposure to sporidesmin and to examine associations between the severity of gross and histological changes to the liver and the activity of gamma-glutamyltransferase (GGT) measured in serum in the sheep at the time of intoxication. METHODS: A group of 50 Romney ewes grazing a mixed ryegrass/white clover pasture were accidentally exposed to sporidesmin for up to 5 weeks. Seventeen sheep showed photosensitisation and four were subject to euthanasia. The remaining sheep were moved to safer pasture and a blood sample collected and analysed for serum GGT activity. The sheep were slaughtered 8 months later. Livers were classified into grossly normal, moderately affected, or severely affected and histology performed to assess portal fibrosis, biliary hyperplasia, portal inflammation, and hepatocellular necrosis. RESULTS: Serum GGT activity ranged from 59 to 1571 IU/L (reference range 32-70 IU/L). Thirteen of the 46 sheep developed clinical signs of facial eczema. However, at slaughter all except four sheep had grossly detectable changes to the shape of the liver including atrophy of the left lobe and the lateral part of the right lobe. Hypertrophy was typically limited to the medial part of the right lobe. In severely affected sheep the liver hypertrophy formed a nodular bulging mass. Changes in the liver shape were classified as severe in 25 and moderate in 17 sheep. Severely affected livers contained significantly more fibrosis than moderately affected livers (p = 0.001, Cliff's delta (d) = 0.68). While there was significantly greater fibrosis and biliary hyperplasia in the left than right lobes, histological changes were present throughout all samples taken of affected livers. Serum GGT activity taken during acute intoxication were correlated to subsequent fibrosis and biliary hyperplasia. CONCLUSIONS: Hepatic fibrosis develops in sheep after a single episode of sporidesmin intoxication, even in sheep with only mildly elevated GGT activity at the time of intoxication. Furthermore, the severity of the subsequent hepatic fibrosis was predicted by the degree of elevation of serum GGT activity during intoxication. CLINICAL RELEVANCE: More research is required to determine how the presence and severity of hepatic fibrosis affect animal production. However, if hepatic fibrosis does decrease production, the consistent development of fibrosis after sporidesmin ingestion reinforces the importance of avoiding exposure of livestock to sporidesmin. ABBREVIATIONS: GGT: Gamma-glutamyltransferase; d: Cliff's delta.

The cellular and molecular toxicity of sporidesmin.

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.

Effects of sporidesmin on cultured biliary tract cells from Romney lambs that differed in their sensitivity to sporidesmin.

AIMS To investigate the effects of sporidesmin on cells cultured from the epithelial surface of sheep gallbladder walls, and to examine cellular responses to sporidesmin using cultures prepared from the gallbladders of sheep from selection lines that differed in sensitivity to sporidesmin-induced liver damage. METHODS Gallbladders were obtained following slaughter of lambs that were selected for resistance or susceptibility to sporidesmin-induced liver damage, or were not selected (controls). Monolayer cell cultures were established after incubation of excised gallbladders with protease to detach the lining epithelial cells from the muscular and connective tissue of the gallbladder wall. Released cells were harvested and transferred to culture flasks or dishes, then incubated with 1 µg/mL sporidesmin and were examined at 5 minute intervals, up to 3 hours, using light microscopy to monitor loss of attachment of cultured cells. Immunofluorescence staining of cell cultures was used to identify cytokeratin 19 as a marker for biliary epithelial cells, and to characterise sporidesmin-induced change in the cellular distribution of actin microfilaments. Gallbladder size was also measured. RESULTS In cultures incubated with sporidesmin, cells at the margins of sheets of cells showed the first signs of change, becoming unanchored from the culture vessels while remaining attached to the cell mass. This was followed by progressive detachment of sheets of cells and clumps of rounded cells. Disruption of cytoplasmic actin microfilaments with accumulation of actin in the cytoplasm adjacent to the plasma membrane preceded major detachment of cells. Cells from susceptible line lambs were extensively rounded up within 1 hour with complete or almost complete detachment within 2 hours, whereas cultures from resistant line lambs generally only contained detaching rounded-up cells at the periphery of monolayers within 2 hours; detachment observed in cells from the control line lambs was intermediate. There was a trend for gallbladders to be smaller in male lambs from the resistant line compared to the control or susceptible lines. CONCLUSIONS Altered cell adhesion and disruption of microfilament actin in biliary cell cultures incubated with sporidesmin suggest that biliary tract pathology may be due to the effects of the toxin on cytoplasmic and cell surface protein networks that affect the integrity of the epithelial lining of the biliary tract. These effects can be interpreted in terms of the hepatobiliary pathology of facial eczema, including potential differences in sensitivity of biliary tract cells that may contribute to inherited resistance and susceptibility to sporidesmin and hence facial eczema.

Effect of a one-off sporidesmin challenge on the milk production of dairy cows.

AIMS To investigate the effects on milk yield in lactating dairy cows of a single dose of sporidesmin, and to categorise the responses based on clinical signs and differing degrees of liver damage, as assessed by activities of γ-glutamyl transferase (GGT) and post-mortem liver histopathology. METHODS Adult lactating dairy cows (n=17) were given a single intra-ruminal dose of 0.24 mg/kg of sporidesmin dissolved in ethanol and diluted in water on Day 0; an additional three cows served as untreated controls. Weekly serum samples were collected between Days -14 and 42 and analysed for activities of GGT. Milk yields were measured daily over the same period. Cows were subjected to euthanasia due to severe clinical signs (n=2) or were slaughtered at the end of the trial. Samples of livers were examined histologically and were scored for lesions on a scale from 0 (normal) to 3 (severe). Based on GGT activities and clinical observations, cows that were treated with sporidesmin were categorised as non-responders (no clinical signs and normal GGT), subclinical (elevated GGT and no clinical signs) or clinical. Outcomes were compared between these three groups and control cows using generalised additive models. RESULTS Seven cows were classified as clinical, and had median liver scores of 22 (95% CI=20.6-23.4), six were subclinical with median liver scores of 8.7 (95% CI=3.8-13.5) and four were non-responders with median liver scores of 2.5 (95% CI=1.2-4.3). Median liver scores for the three control cows were 1 (95% CI=-0.8-2.1). Activities of GGT increased in subclinical and clinical cows around Day 7. The milk yield of all cows treated with sporidesmin, including non-responder cows, started to decrease on Day 1, and reached a nadir (a drop of between 9 and 85%) on Day 7. CONCLUSIONS AND CLINICAL RELEVANCE It is likely that the overall effects of sporidesmin consumption on milk production by the national herd in New Zealand are hugely underestimated, especially considering its effects on non-responder and subclinical cows as shown in this trial. In view of the results presented here, the authors are suggesting a change to the definition of response to sporidesmin from non-responder, subclinical, and clinical, to subclinical-low, subclinical-high, and clinical, when measuring a combination of GGT activities, clinical signs and milk yields during facial eczema-risk seasons (summer-autumn).

Variability in measurement of Pithomyces chartarum spore counts.

AIMS To examine the agreement between spore counts of Pithomyces chartarum measured in a single aliquot of wash water with counts from multiple aliquots from the same 60 g pasture sample, and between spore counts measured in an individual 60 g pasture sample with counts from three 60 g pasture samples selected from the same 200 g paddock sample. MATERIALS AND METHODS Four Waikato dairy farms were visited once weekly from early January to late May 2013. One paddock, with 40 sampling sites, was selected per farm. At each visit, ∼200 g of pasture was collected per site. Spore counting was undertaken using a standard method, except that three separate 60 g pasture samples per 200 g paddock sample was counted; and for each 60 g pasture sample, spores were counted in 10 aliquots of wash water. The relationship between the results of a single aliquot and 6-10 aliquots of wash water from the same 60 g grass sample were assessed by calculating 95% prediction intervals. Limits of agreement analysis was used to assess the agreement between counts from one, two or three aliquots per 60 g pasture sample compared with 10 aliquots, and between counts from one and three 60 g pasture samples from the same 200 g paddock sample. RESULTS Comparing spore counts from individual aliquots with multiple aliquots resulted in large prediction intervals and 95% limits of agreement, which increased with increasing spore count. For an individual aliquot count of 2 spores, the 95% prediction interval for the count from 10 aliquots was 3-49 spores, and for an individual count of 10 spores the 95% prediction interval was 28-222 spores. Increasing the number of aliquots counted improved agreement. For a total count of 10 spores measured in 10 aliquots, the 95% limits of agreement, based on a single aliquot, were 2-50 spores, and for three aliquots were 5-20 spores. The agreement in spore counts measured in one compared with three 60 g pasture samples was moderate and also decreased with increasing spore count; the 95% limits of agreement were 4-14.5 for a mean spore count of 10. CONCLUSIONS AND CLINICAL RELEVANCE Measuring the spore counts of three aliquots of wash water per 60 g grass sample improved repeatability, and should be used as the standard technique, particularly when determining whether to start or finish facial eczema control programmes.

Both canonical and noncanonical Wnt signalling may be required for detoxification following ETP class mycotoxin exposure.

Fungal infections (mycotoxicoses) are a growing global threat for both health and food production, and the available tools for effective detection, monitoring and treatment remain limited. Mycotoxins of the so-called ETP class can cause disease in humans (notably immunocompromised clinical patients) and otherwise healthy ruminant production animals. Understanding the molecular responses caused by ETP toxicity responses will inform diagnostics and guide possible interventions. Here we provide empirical evidence that exposure of hepatic cells to the ETP mycotoxin Sporidesmin A may trigger both canonical and noncanonical Wnt signalling, mediated through miRNA regulation, and regulate the particular expression of CYP2C family members. These data suggest cellular adaptation to mycotoxin exposure is an epigenetically dependent process leading to the co-ordination of multiple Wnt pathways to drive appropriate downstream detoxification mechanisms.

Facial eczema management protocols used on dairy farms in the North Island of New Zealand and associated concentrations of zinc in serum.

AIMS: To describe and evaluate the current practices used to manage and prevent facial eczema (FE) in North Island dairy herds, and determine the within-herd prevalence of cows with elevated activities of gamma glutamyl transferase (GGT), and with concentrations of Zn in serum <18 µmol/L. METHODS: Between January and May 2014, 105 herd managers from throughout the North Island of New Zealand were invited to participate in the study when regional spore counts for Pithomyces chartarum started to rise towards 30,000 spores/g pasture. Managers selected 10 representative cattle that were weighed and blood-sampled by the herd veterinarian. Blood samples were analysed for concentrations of Zn in serum and GGT activity. Pasture samples were also collected and submitted for spore count estimation. Finally a survey of farm management practices relating to prevention of FE was completed by the herd manager. A mixed-effects logistic regression model was used to determine associations between herd-level and cow-level explanatory variables and the probability of a cow having a concentration of Zn in serum <18 µmol/L. RESULTS: Of the 1,071 cows tested, 79 (7.3 (95% CI=5.8-9.0)%) had GGT activity in serum >300 IU/L, and 35/106 (33 (95% CI=24.2-42.8)%) herds had ≥1 of the 10 cows sampled with GGT activity >300 IU/L. Of the 911 cows that were being treated with Zn, concentrations of Zn were between 18-35 µmol/L in 398 (43.6 (95% CI=40.4-46.9)%) cows, were >35 µmol/L in 32 (3.5 (95% CI=2.4-4.1)%) cows, and <18 µmol/L in 479 (52.6 (95% CI=49.3-55.9)%) cows. After adjusting for the confounding effect of region, the odds of a cow having concentrations of Zn in serum <18 µmol/L were 5.5 (95% CI=1.1-29) times greater for cows supplemented with zinc in water compared with those supplemented by drenching. Of the 105 herd managers, 103 (98%) stated that they had access to regional spore count data, but only 35/105 (33%) reported that they measured spore counts on their own farm. Overall, 98/105 (93%) managers reported that they had some form of FE management programme in place. Fungicides were used on their own or in combination with zinc treatments in 10 herds, ZnSO4 in water troughs was used in 68 herds, oral drenching with ZnO in nine herds, and ZnO supplied in-feed in 26 herds. Estimated daily dose rates of zinc were less than that required to treat a 400 kg cow on 42/68 farms that administered ZnSO4 in the water or ZnO as a drench. CONCLUSION AND CLINICAL RELEVANCE: This study has shown that FE management on dairy farms in the North Island of New Zealand could be substantially improved. It is likely that improved FE management would occur if herd managers were provided with more feedback on the success (or otherwise) of their FE management programmes.

Bile duct lesions associated with turnip (Brassica rapa) photosensitization compared with those due to sporidesmin toxicosis in dairy cows.

Cattle grazing turnips or other brassica forage crops occasionally develop hepatogenous photosensitization. In New Zealand, cases of bovine photosensitization associated with such crops frequently occur during late summer and fall, and this coincides with the facial eczema (sporidesmin toxicosis) "season." Clinical chemistry findings in acute photosensitization cases associated with both brassica and facial eczema include marked serum elevations in γ-glutamyl transferase and glutamate dehydrogenase activities. Distinctive bile duct lesions of "subacute" turnip photosensitization in 2 cows, comprising microscopic cholangiectasis with concentric periductal fibrosis of small bile ducts, and a spectrum of changes from bile duct necrosis progressing to obliterative sclerosis are described. These bile duct lesions are compared with those in 3 cases of "subacute" facial eczema in adult cows, where medium-sized and larger ducts tend to be involved and bile duct hyperplasia and portal fibrosis are more prominent, often leading to bridging between neighboring portal triads.

Clinicopathological studies on facial eczema outbreak in sheep in Southwest Turkey.

After very hot summer, 22 sheep from 5 different flocks consisting of approximately 150-200 animals each were diagnosed with facial eczema in September 2005, in southwest Turkey. Photophobia, corneal opacity, severe ulcers of the facial skin, especially localized around the eyes and mouth, and 3% mortality were the most prominent clinical symptoms. GGT levels of the animals were very high and varying between 261- 328 U/l. While the activities of ALT and total bilirubin were elevated and AST was normal in affected sheep. Total bilirubin level was higher than normal. Seven of the 22 sheep were euthanatized and necropsy was performed on all of these animals. Severe icterus, hepatomegaly, enlarged gallbladder, congestion of mesenteric vessels were the common necropsy findings. Histopathological changes of the liver included necrosis of the hepatocytes, cholangiohepatitis characterized by mononuclear inflammatory cell infiltrate in the portal area and mild to severe fibrosis around bile ducts. A diagnosis of sporidesmin toxicosis was made based on the histopathology of the livers, the elevation in liver enzymes, and the development of cutaneous lesions consistent with photosensitization and high spore counts in the ruminal contents. Surviving sheep were treated with procaine penicillin + dihidrostreptomycin sulfate, multivitamin complexes and flunixin meglumine. Additionally, zinc sulphate was also given at a dose of 6 gr per 100 lt drinking water for 28 days. All treated sheep recovered. Pasture spore counts were between 96,300- 267,500 spores/g grass.

Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones.

Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time- and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogues.

[First case of pithomycotoxicosis (facial eczema) in the Netherlands]. / Pithomycotoxicosis of facial eczema bij het rund voor de eerste maal aangetoond in Nederland. Zonnebrand als koppelprobleem op rundveebedryjven.

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.

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.

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.

Zinc protection of HepG2 cells from sporidesmin toxicity does not require de novo gene transcription.

Sporidesmin is an epidithiodioxopiperazine mycotoxin secreted by the saprophytic fungus Pithomyces chartarum. Ingestion of sporidesmin by ruminants grazing on the saprophyte infested pasture causes severe liver and bile duct damage leading to secondary photosensitisation. Zinc supplementation is used as an effective prophylaxis against sporidesmin toxicity in ruminants, however, the mechanism by which zinc protects is unknown. This study used the human hepatoma cell line, HepG2, as a model to examine the mechanism of zinc protection against sporidesmin toxicity. Treatment of cells with various concentrations of sporidesmin (0-10 microg/ml) resulted in a sigmoidal dose response curve with an LC50 of 5 microg/ml. Cells were protected from sporidesmin toxicity by pre-treatment for 2h or 16 h with zinc sulphate in a concentration dependent manner, with significant protection at 50 microM zinc and maximal protection at 200 microM zinc. To determine whether zinc protection required de novo gene transcription, cells were treated with the transcriptional inhibitor actinomycin D for one hour prior to and throughout the zinc pre-treatment. The presence of actinomycin D did not significantly reduce the zinc protection against sporidesmin cytotoxicity (80% protection without actinomycin D versus 71% protection with actinomycin D). Therefore, de novo gene transcription does not play a major role in the mechanism of zinc protection against sporidesmin toxicity in HepG2 cells.

Metal complexes of the mycotoxins sporidesmin A and gliotoxin, investigated by electrospray ionisation mass spectrometry.

The mycotoxin sporidesmin A (spdA), responsible for the intoxication of animals, causing facial eczema, has been investigated by electrospray ionisation mass spectrometry. Protonated [spdA+H](+) and deprotonated [spdA-H](-) ions are observed in positive and negative ion modes respectively. Reduced spdA, formed by cleavage of the disulfide bond by Na[BH(4)] gives an ion [spdA+H](-), and forms ions of the type [2spdA+M](2-) with a range of divalent metal ions M(2+)=Zn(2+), Cd(2+), Hg(2+), Sn(2+) and Fe(2+). Sodium-containing analogues [2spdA+M+Na](-) are observed, particularly at high cone voltages, where they are stable towards cone voltage-induced fragmentation, indicating appreciable stability of the (spdA)(2)M system. A competition experiment between Cd(2+) and Zn(2+) demonstrates that reduced spdA has a higher affinity for Cd(2+) ions. The related gliotoxin (gtx) forms analogous [2gtx+M](2-) and [2gtx+M+Na](-) ions. The reduction and metal complexation of spdA can be monitored by (1)H NMR spectroscopy, and results in chemical shift changes for those protons adjacent to the sulfur atoms. The isolation of a polymeric cadmium-spdA complex is also reported.