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.     

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 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.     

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.     

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.     

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.     

Pathological lesions following an experimental intoxication with aflatoxin B1 in broiler chickens

A follow-up of chickens dosed orally over 21 days with 0.2 and 3 micrograms of aflatoxin B1 (AFB1) g-1 of bodyweight daily and their subsequent recovery 10 days after withdrawal of contaminated food was conducted. Vacuolation of liver cells during the initial days of the intoxication and cellular depletion in the follicle medulla of the bursa of Fabricius were the lesions which appeared first and persisted during the recovery phase in both groups of intoxicated animals. The intensity of these lesions and their persistence was related to the dose of aflatoxin ingested. A significant reduction in the bodyweight and absolute weights of liver, bursa of Fabricius spleen and thyroid was observed in the higher dose group.     

A technique for the quantification of Duddingtonia flagrans chlamydospores in sheep faeces

Previous observations showed that Duddingtonia flagrans chlamydospores were visualized in McMaster chambers containing faeces of treated sheep. This trial explored the McMaster technique as a tool to quantify chlamydospores in sheep faeces. A range of individual chlamydospore doses (from 19.5 x 10(6) to 177.5 x 10(6)) were offered orally to nine lambs for 7 consecutive days. A faecal sample (5 g) was daily obtained from the rectum of each animal (from days 1 to 13) to perform the McMaster technique using a sugar flotation fluid with 1.27 g/mL density. Each chlamydospore counted in the McMaster chamber was considered as 50 chlamydospores per g of faeces (CPG). The results confirmed that the estimated CPG was associated with the daily dose offered to the animals (r(2)=0.90; P<0.001). Furthermore, the total chlamydospore dose received by each animal was strongly associated to the total quantity of CPG obtained from the bulk faeces (TCtot) (r(2)=0.96; P<0.0001). Quantification of CPG can be used as a helpful tool to determine the number of chlamydospores reaching the faeces in orally dosed animals. This could be used to evaluate the efficacy of D. flagrans for the control of gastrointestinal nematode larvae in sheep faeces.     

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.     

Furosemide for prevention of cyclophosphamide‐associated sterile haemorrhagic cystitis in dogs receiving metronomic low‐dose oral cyclophosphamide

Sterile haemorrhagic cystitis (SHC) is a known risk of cyclophosphamide treatment. Diuresis using furosemide is effective in canines when maximally tolerated dosed cyclophosphamide is administered. This retrospective study aimed to determine whether orally administered furosemide decreased the incidence of SHC. Secondary aims were to identify predisposing factors for SHC. One‐hundred and fifteen dogs treated with metronomic cyclophosphamide were analysed retrospectively. Populations were not randomized. 25 dogs (21.7%) developed SHC. Furosemide administration significantly reduced the likelihood of SHC development (P = 0.010, where SHC was diagnosed in 30.3% of dogs administered cyclophosphamide without furosemide, and 10.2% of dogs administered cyclophosphamide with furosemide). Age, gender, breed, bodyweight, number of cyclophosphamide treatments, piroxicam use and previous or pre‐existing disease were not found to be associated with SHC development. This study demonstrates furosemide is effective in the prevention of SHC and its use may be considered when implementing metronomic cyclophosphamide therapy.     

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.     

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.     

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.     

Evaluation of the toxicity of Adonis aestivalis in sheep

To determine the toxicity of Adonis aestivalis (adonis) in sheep, adult Suffolk ewes were administered 1 per cent bodyweight adonis via surgically placed rumen cannulas in an acute, high-dose toxicity study, and 0.2 per cent bodyweight daily in a two-week, low-dose toxicity study. The ewes received cardiac examinations before dosing, 24 and 48 hours after dosing with 1 per cent bodyweight adonis, and after continuous low-dose administration. All the ewes administered adonis had transient sinus arrhythmias after receiving 1 per cent bodyweight adonis. Two of the three ewes had transient reduced fractional shortening after administration with 1 per cent bodyweight adonis; the same two ewes had reduced fractional shortening after the low-dose treatment regimen. No gross or microscopic lesions were seen when the ewes were examined postmortem at the end of the study.     

Target animal safety and tolerance study of pyrantel pamoate paste (19.13% w/w pyrantel base) administered orally to horses

Pyrantel pamoate paste (19.13% w/w pyrantel base) for the treatment of tapeworm, Anoplocephala spp was evaluated for target animal safety and tolerance in horses treated orally at 0, 1, 3, 5, and 10 times the clinical dose of 13.2 mg pyrantel base/kg body weight administered daily for six consecutive days. Parameters evaluated included clinical signs, food and water consumption, body weights, physical examinations, clinical pathology (hematology, coagulation, serum chemistry, urinalyses, and fecal examinations), complete necropsy, organ weights, and histopathology. No adverse events or test article-related effects were observed in any treatment group during daily clinical observations of the test animals. Statistically significant changes (P < .05) lacked a dose- and/or time-dependent trend and were considered incidental. Administration of pyrantel pamoate paste did not produce any macroscopic or microscopic tissue effects in any dose group of either sex. The no-observed-effect-level (NOEL) for pyrantel pamoate paste, when administered orally to horses once daily for 6 consecutive days, was determined to be 132 mg/kg/day. Pyrantel pamoate paste (19.13% w/w pyrantel base) can be safely administered orally to horses at 13.2 mg of pyrantel base/kg for the treatment of Anoplocephala infestations.     

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.     

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.     

An evaluation of the efficacy of netobimin against Dicrocoelium dendriticum in sheep

A trial was carried out to assess the efficacy of a nitrophenylguanidine compound, netobimin against Dicrocoelium dendriticum in naturally infected sheep. At a dose rate of 20 mg/kg bodyweight administered orally the drug was highly effective, producing a mean reduction of 98.9 per cent in the fluke burdens of treated animals compared with untreated controls. No side effects were observed in the treated sheep.     

Plasma kinetics and efficacy of oral megazol treatment in Trypanosoma brucei brucei-infected sheep

Experimentally infected sheep have been previously developed as an animal model of trypanosomosis. We used this model to test the efficacy of megazol on eleven Trypanosoma brucei brucei-infected sheep. When parasites were found in blood on day 11 post-infection, megazol was orally administered at a single dose of 40 or 80mg/kg. After a transient aparasitaemic period, all animals except two relapsed starting at day 2 post-treatment, which were considerated as cured on day 150 post-treatment and showed no relapse after a follow-up period of 270 days. In order to understand the high failure of megazol treatment to cure animals, a kinetic study was carried out. Plasma concentrations of megazol determined, by reverse-phase high-performance liquid chromatography at 8h post-treatment in these animals, were lowered, suggesting slow megazol absorption, except in cured animals. However, megazol plasma profiles in uninfected sheep after a single oral dose of megazol showed a fast megazol lowered absorption associated with a short plasma half-life of drug. Inter-individual variation of megazol pharmacokinetic properties was also observed. These findings suggested that the high failure rates of megazol treatment were related to poor drug availability after oral administration in sheep. In conclusion, megazol could cure sheep with T. b. brucei infection but oral administration was not an effective route.     

天然植物制剂“开口健”对小鼠免疫功能影响

目的:探讨天然植物制剂"开口健"对实验小鼠吞噬功能以及抗体产生能力的影响。方法:每天用不同剂量(0.125、0.25、0.5g)"开口健"给雄性ICR小鼠灌胃,用生理盐水作为空白对照。连续灌胃10d后,检测小鼠的体重变化和脾脏指数,用碳粒廓清法检测小鼠巨噬细胞吞噬功能,用溶血素分光光度法检测小鼠抗体产生水平。结果:与生理盐水组相比,高、中、低剂量"开口健"灌胃组小鼠的体重均有提高,中、低剂量组提高显著(P0.05);各试验组脾脏指数均有增加,但统计分析差异不显著(P0.05);各试验组小鼠巨噬细胞吞噬指数和廓清指数均有显著提高(P0.05);各试验组小鼠抗体水平均有极显著上升(P0.01)。结论:"开口健"能通过提高小鼠单核巨噬细胞碳廓清能力和促进抗体产生而提高小鼠免疫功能。