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.     

WHITE LIVER DISEASE OF SHEEP

SUMMARY Outbreaks of ovine white liver disease (WLD) on 7 farms in eastern Victoria were investigated. Most occurred in late spring and mainly affected lambs 3 to 6 months old, with a morbidity of 20 to 100% and mortality of 8 to 15%. Clinically affected lambs showed illthrift, emaciation and bilateral, serous, ocular discharge. Clinical pathology showed mild anaemia, elevated serum liver enzymes (GGT, OCT, AST) and low levels of serum vitamin B12. Grossly, the livers were pale, fatty and friable; microscopically there was parenchymal fatty change, bile duct proliferation and ceroid pigmentation. Liver cobalt values were consistently low (mean 0.4 ± 0.4 μmol/kg D.W.). Levels of cobalt in pasture from 2 properties were very low (0.34 μmol/kg D.W.). The diagnosis of white liver disease was made on the basis of clinical features, specific liver pathology and low cobalt status. Treatment trials established that cobalt injections or oral bullet administration resulted in clinical improvement, significant weight gains, and improved serum vitamin B12 levels. WLD did not recur in previously affected sheep using these treatments. However, when blocks containing cobalt were available continuously, WLD recurred 2 years after the initial outbreak.     

A comparison of vitamin B12 levels in the liver and serum of sheep receiving treatments used to correct cobalt deficiency

The efficacies of four methods, used for the prophylaxis of cobalt deficiency in sheep as measured by the elevation of liver and serum vitamin B12 levels, were compared in marginally deficient sheep over 14 weeks. The methods used were weekly drenches of either cobalt sulphate or cobalt chelate (EDTA) three-weekly injections of hydroxocobalamin, and ruminal cobalt pellets. On the basis of elevated liver and serum vitamin B12 levels, chelated cobalt was shown to be available to rumen microflora for the synthesis of vitamin B12. However, at no stage were liver and serum vitamin B12 levels of sheep receiving the chelate significantly different from those receiving the same amount of cobalt as the sulphate. After five, three-weekly injections of hydroxocabalamin liver vitamin B12 levels were significantly higher (p<0.01) than for the other treatments, with the exception of cobalt sulphate. Cobalt pellets led to an initial rapid and significant rise in serum vitamin B12 when compared with the other treatments. However, at four weeks there was no significant difference between treatment groups for serum vitamin B12. Fourteen weeks after the administration of cobalt pellets, serum and liver.vitamin B12 levels in this group were not significantly different from those of untreated sheep. At this time, three out of 12 sheep had lost their pellets.     

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.     

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.     

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.     

A comparison of vitamin B12 levels in the liver and serum of sheep receiving treatments used to correct cobalt deficiency

The efficacies of four methods, used for the prophylaxis of cobalt deficiency in sheep as measured by the elevation of liver and serum vitamin B₁₂ levels, were compared in marginally deficient sheep over 14 weeks. The methods used were weekly drenches of either cobalt sulphate or cobalt chelate (EDTA); three-weekly injections of hydroxocobalamin, and ruminal cobalt pellets.

On the basis of elevated liver and serum vitamin B₁₂ levels, chelated cobalt was shown to be available to rumen microflora for the synthesis of vitamin B12. However, at no stage were liver and serum vitamin B₁₂ levels of sheep receiving the chelate significantly different from those receiving the same amount of cobalt as the sulphate.

After five, three-weekly injections of hydroxocabalamin liver vitamin B₁₂ levels were significantly higher (p < 0.01) than for the other treatments, with the exception of cobalt sulphate.

Cobalt pellets led to an initial rapid and significant rise in serum vitamin B₁₂ when compared with the other treatments. However, at four weeks there was no significant difference between treatment groups for serum vitamin B₁₂. Fourteen weeks after the administration of cobalt pellets, serum and liver. vitamin B₁₂ levels in this group were not significantly different from those of untreated sheep. At this time, three out of 12 sheep had lost their pellets.     

Reference curves to diagnose cobalt deficiency in sheep using liver and serum vitamin B12 levels

Reference curves demonstrating the relationship between serum or liver vitamin B12 and weight gain were derived from the examination of 16 published and 48 unpublished N.Z. trials. From these curves probability of obtaining an economic reponse (>10g/day body weight increase) for any serum or liver vitamin B12 can be determined. No significant (P<0.05) weight gain responses occurred to vitamin B12 or cobalt treatment in trials with mean serum vitamin B12 levels above 500 pmol/l or liver vitamin B12 levels greater than 500 nmol/kg. The reference curves were therefore derived from trials with vitamin B12 levels below these levels; 36 trials with serum vitamin B12 and 19 trials with liver vitamin B12 data. The mean vitamin B12 level at the mid point of the weight gain response period was selected from each trial. Examination of serum vitamin B12 reference curves for spring, summer, autumn and winter indicated that curves derived from data closest to the middle of January (summer) adequately reflected response to treatment at any time during the first year of life. Reference curves for liver vitamin B12 also used data closest to middle of January. This was partly because insufficient liver data was available to compare seasonal variations. The fitted response curve approached 0 gram/day at 500 pmol/l for serum vitamin B12 and 375 nmol/kg for liver vitamin B12. The minimum vitamin B12 level at which an economic response to treatment (>10 g/day) is not likely was 336 pmol/l for serum and 282 nmol/kg for liver.     

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.     

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.     

Caprine hepatic lipidosis induced through the intake of low levels of dietary cobalt

Forty-one, 10-week-old newly weaned goats were randomly allocated into two groups, namely control (n=22) and treated (n=19). Kids in both groups were fed Rhodegrass hay ad libitum that contained < 0.1 mg/kg DM cobalt and 150 g/day of a commercially prepared ruminant concentrate that contained approximately 0.12 mg/kg DM cobalt. This diet provided the minimum daily requirement of cobalt as specified for sheep. The treated goats were supplemented with bi-monthly subcutaneous injections of 2000 microg of hydroxycobalamin. All goats were weighed and blood samples collected monthly for haematological, clinical biochemical and serum vitamin B12 analysis. After a 10-month experimental period the goats were slaughtered. The control animals exhibited significantly (P<0.05) lower weight gains, and had dry scruffy hair coats. In addition, there was a decline in erythrocyte counts, mean haemoglobin, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration. Controls also exhibited significantly (P<0.05) lower levels of total serum proteins and elevated levels of serum alkaline phosphatase compared to treated goats. Fourteen (63.6%) of the control goats developed pathology consistent with reported field cases of hepatic lipidosis associated with low liver levels of cobalt. Only one (5.3%) of the treated goats developed hepatic lipidosis. Contrary to previous reports that suggested that goats are less sensitive to low levels of dietary cobalt than sheep, it is apparent that this is not the case with Omani goats. This is the first report of the induction of hepatic lipidosis in goats due to feeding low levels of cobalt in their diet.     

Evaluation of intraruminal devices for combined facial eczema control and trace element supplementation in sheep

Intraruminal devices containing zinc oxide are very effective in preventing facial eczema in sheep. In the present study such devices augmented with various amounts of selenium and cobalt have been evaluated for their ability to improve the Se and CO status of pregnant ewes, as reflected by changes in blood Se and serum Vitamin B12 concentrations. Devices containing 16.4 mg of Se (as sodium selenate) and 20.4 mg of Co (as cobalt sulphate) were effective in increasing and maintaining elevated blood Se concentrations for at least 84 days and serum Vitamin B12 for 42 days. Such devices will therefore prevent trace element deficiencies in sheep as well as providing protection against facial eczema.     

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.     

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.     

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.     

Absence of a weight gain response to Vitamin B12 supplementation in weaned dairy heifers grazing pastures of marginal cobalt content

AIM: To obtain information on serum and liver vitamin B12 and urinary methylmalonic acid concentrations as diagnostic tests to predict a weight gain response to supplementation with vitamin B12 in young dairy cattle when grazing pasture of low cobalt content. Methodology. Forty dairy cattle (12 Friesian, 14 Friesian x Jersey and 14 Jersey) were allocated to two equal sized groups, treated and untreated, based on liveweight. At monthly intervals for 14 months, all animals were weighed, their serum and urine sampled, their liver biopsied and the pasture sampled from the paddocks they were grazing and going to graze. Serum and liver were assayed for vitamin B12 concentrations. For the first 5 months of the trial, urine was assayed for methylmalonic acid concentrations. Both washed and unwashed pasture samples were assayed for cobalt concentrations. RESULTS: No weight gain response occurred vitamin B12 supplementation in young growing cattle grazing pasture with a cobalt concentration of 0.04-0.06 mg/kg DM. For 5 months of the trial, liver vitamin B12 concentrations from untreated calves were in the range 75-220 nmol/kg and serum vitamin B12 concentrations were as low as 72 pmol/1. There was no associated growth response to supplementation. CONCLUSION: Further trials involving young cattle grazing pastures with cobalt concentrations less than 0.04 mg/kg DM are required to reliably determine liver and serum vitamin B12 concentrations at which growth responses to vitamin B12 or cobalt supplementation are likely under New Zealand pastoral grazing conditions.     

Effect of ingestion of soil on the iodine, copper, cobalt (vitamin B12) and selenium status of grazing sheep

AIM: To determine the impact of ingestion of soil on the iodine (I), selenium (Se), copper (Cu) and cobalt (Co; vitamin B12) status of young sheep. METHODS: Twenty young sheep were divided into two groups; one group was fed lucerne pellets, while the other group was fed lucerne pellets plus 100 g soil, for 63 days. At the end of the study the animals were blood-sampled, slaughtered, and the liver removed, and concentrations of I, Cu, vitamin B12 and Se were determined. RESULTS: The ingestion of soil significantly increased concentrations of I and vitamin B12 in serum, but had no effect on concentrations of Cu and Se in serum/blood and liver, and vitamin B12 in liver. CONCLUSION: Ingested soil can be a significant source of I and Co (vitamin B12) for grazing sheep.     

Changes in serum concentrations of methylmalonic acid and vitamin B12 in cobalt-supplemented ewes and their lambs on two cobalt-deficient properties

AIM: To determine concurrent changes in serum methylmalonic acid (MMA) and vitamin B12 concentrations of ewes and their lambs on cobalt-deficient properties, subsequent to cobalt supplementation. METHODS: Three experiments were carried out on two farms. Groups of ewes (n=25-50) were either supplemented with cobalt bullets during late pregnancy, 23-47 days before the mean lambing date, or left unsupplemented. In two experiments, lambs from within each group were supplemented directly by vitamin B12 injection at 3-weekly intervals from birth, and in the third experiment by injection with micro-encapsulated vitamin B12 at tailing and 3 months later. Pasture samples were obtained for analysis of cobalt content at each sampling time. Blood samples were obtained and liveweight recorded from ewes and lambs at approximately monthly intervals. On one farm (two experiments), liver and milk samples were obtained from ewes and liver samples from lambs. RESULTS: Serum vitamin B12 concentrations in unsupplemented ewes fell below 250 pmol/L during early lactation in all experiments and mean concentrations as low as 100 pmol/L were recorded. MMA concentration was maintained below 2 micromol/L in serum from supplemented ewes but increased to mean concentrations ranging from 7 to 14 micromol/L at the nadir of serum vitamin B12 concentration during peak lactation. A significant liveweight response to supplementation was recorded in ewes on one property, and the vitamin B12 concentration in the ewes' milk and in the livers of their lambs more than doubled. No liveweight-gain response to supplementation was observed in lambs on this property. Mean serum MMA concentrations in lambs ranged from <2 in supplemented, to 19.2 micromol/L in unsupplemented lambs, and the latter had concurrent serum vitamin B12 concentrations of >300 pmol/L. Pasture cobalt concentration was lowest at 0.04-0.09 microg/kg dry matter (DM) on the property on which responses in lambs occurred but considerably higher (>0.09 microg/kg DM) on the property on which responses in ewes occurred. On the second property, serum vitamin B12 concentrations in lambs at tailing were extremely low (100 pmol/L), irrespective of supplementation of dams with cobalt. Mean serum MMA concentration was increased to 20 and 42 micromol/L in lambs from supplemented and non-supplemented ewes, respectively. Weight-gain response to direct supplementation of lambs with vitamin B12 occurred during suckling in the latter, but not the former. Lambs from ewes supplemented with vitamin B12 showed a much bigger increase in serum vitamin B12 concentrations a month after supplementation than did lambs from unsupplemented ewes (+1,400 pmol/L vs + 650 pmol/L). CONCLUSIONS: Serum MMA concentration gave a more precise indication of responsiveness to vitamin B12 or cobalt supplementation than serum vitamin B12 concentrations in ewes and lambs. Neither very low serum vitamin B12 nor elevated MMA concentrations were necessarily indicative of responsiveness to supplementation in suckling lambs, but the latter gave an early indication of impending responsiveness. Supplementation of the ewe with a cobalt bullet appeared to protect the growth performance of the lamb for 90 days and influence the subsequent serum vitamin B12 response in the lamb to vitamin B12 supplementation. CLINICAL SIGNIFICANCE: Supplementing ewes with cobalt bullets in late pregnancy can improve the vitamin B12 status of their lambs, and modify their response to vitamin B12 supplementation.     

An assessment of the efficacy and safety of selenium and cobalt included in an anthelmintic for sheep

A trial was devised to assess whether the administration of selenium and cobalt together with the anthelmintic mebendazole (Ovitelmin S&C) was safe and could improve the supplies of selenium and cobalt for adult sheep fed a whole grain diet, low in both elements, which produced a steady decrease in blood glutathione peroxidase (GSHPx) and plasma vitamin B12 concentrations. Ovitelmin S&C, when given orally in a single dose as a suspension containing 0.34 mg selenium/ml, and 0.44 mg cobalt/ml (to provide 0.11 mg selenium and 0.15 mg cobalt/kg liveweight) significantly increased the GSHPx activity in blood. After a second dose given 28 days later the rate of change increased from 2.5 to 3.5 u/g haemoglobin/day. The responses in GSHPx were similar for a preparation which contained twice the concentration of selenium. Ovitelmin S&C increased the concentration of vitamin B12 in the plasma by about 1000 pg/ml for four to seven days after each dose and the increases were similar to those observed in sheep treated with an Ovitelmin preparation containing 45 times more cobalt (providing 6.7 mg cobalt/kg liveweight). After 63 days, liver vitamin B12 concentrations were 43 per cent higher in the cobalt treated than in the untreated groups (P less than 0.01) with no differences among the groups given cobalt. Neither adverse reactions nor signs of toxicity followed the administration of Ovitelmin S&C or Ovitelmin containing the higher concentrations of selenium and cobalt.     

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.