Sapogenin levels in Narthecium ossifragum plants and Ovis aries lamb faeces during two alveld outbreaks in Møre og Romsdal, Norway, 2001.

The proposal that saponins produced by the lily bog asphodel (Narthecium ossifragum) may be the direct cause of the hepatogenous photosensitization disease alveld seen in Norwegian lambs was investigated by comparing sapogenin levels in two control and two toxic pastures, and in faeces from lambs grazing the four pastures in the Halsa and Surnadal municipalities, Møre og Romsdal county, Norway. Generally similar levels of sapogenins, determined after hydrolysis of parent plant saponins, were found in Narthecium leaves collected in June/July 2001 from the two alveld outbreak areas and two nearby control areas. Differences in the median sapogenin levels determined for leaf samples in outbreak and control areas were not statistically significant. The total level of free and conjugated sapogenins in faeces recovered from the rectums of lambs grazing the outbreak and control pastures areas varied greatly. The results obtained do not support the hypothesis that a dose-response relationship exists between Narthecium saponin levels and the occurrence of alveld outbreaks.

Pesticide residues in adipose tissue from hippopotami (Hippopotamus amphibius L) living in and adjacent to the Luangwa River in Zambia.

The concentration of organochlorines (OCs) such as organochlorine pesticides and polychlorinated biphenyls were measured in adipose tissue collected from 14 male hippopotami at Mfuwe in the southern part of the Luangwa National Park, Zambia. The samples contained low levels of OCs, and the concentrations of OCs were comparable to or lower than reported for wild herbivores studied in other parts of the world.

A simple gag for the intragastric dosing of guinea pigs (Cavia porcellus).

A simple gag has been designed and tested which enables the intragastric intubation of the guinea pig by one person without the use of sedation or light anaesthesia. The system described was used successfully for the daily dosing of materials to 7 guinea-pigs for 14 days without discomfort, aspiration or any injury to the upper gastro-intestinal tract.

Experimental Narthecium ossifragum nephrotoxicity in cervids from Norway.

One moose (Alces alces), two red deer (Cervus elaphus), two reindeer (Rangifer tarandus) and two fallow deer (Dama dama) were dosed intraruminally with an aqueous extract made from 30 g of bog asphodel (Narthecium ossifragum) (wet weight) per kg live weight. The moose and one of the two reindeer were mildly depressed and had reduced appetite 3 to 7 days and 1 to 4 days after dosing, respectively. The serum creatinine and urea concentrations increased markedly in the moose and red deer, and moderately in the reindeer. No increase in serum creatinine and urea was observed in the fallow deer. Histopathological examination of the kidneys of the animals, killed 8 to 10 days after dosing, revealed tubular epithelial cell degeneration, necrosis, and regeneration in the moose, red deer and reindeer. The renal lesions were severe in the moose, moderate in the red deer and mild in the reindeer. No histopathological lesions were seen in the kidneys of the fallow deer.

Hepatic and renal concentrations of copper and other trace elements in hippopotami (Hippopotamus amphibius L) living in and adjacent to the Kafue and Luangwa Rivers in Zambia.

Hepatic and renal concentrations of the elements arsenic, cadmium, cobalt, copper, lead, manganese, mercury, molybdenum, selenium and zinc were studied in samples collected from hippopotami from the Kafue River in the Kafue National Park and the Luangwa River in the Southern Luangwa National Park in Zambia. There were no significant differences between trace element concentrations in the tissues of the hippopotami taken in the Kafue River and the Luangwa River. The concentrations of copper and other essential elements were similar to those reported in normal domestic and wild ruminants. Judging by the results obtained in this study, pollution from the mining activity around the Kafue River drainage area in the Copperbelt region has not led to any accumulation of elements in tissues of the hippopotami in the Kafue National Park. The trace element concentrations observed may serve as reference for similar future studies on hippopotami.

Seasonal variation in cotyledoside concentration of Tylecodon wallichii (Harv.) Tölken subsp. wallichii sampled in a krimpsiekte-prevalent region.

Krimpsiekte, an economically important neuromuscular affliction of small stock, follows upon ingestion of certain members of the Crassulaceae (plakkies) containing cumulative neurotoxic bufadienolides. Tylecodon wallichii (Harv.) Tölken subsp. wallichii is probably the most important species of the group of plants causing krimpsiekte. The growing tip of the stem and various other plant parts of T. wallichii, when available, were collected monthly. The seasonal variation in cotyledoside content of the plant was measured. Cotyledoside concentration was determined by high performance liquid chromatographic-electrospray mass spectrometry analysis (HPLC-ESMS). The cotyledoside concentration in the plant stems fluctuated substantially during the year, but tended to be higher in the cold winter months and increased again in the spring and early summer. Elevated plant stem concentrations corresponded with natural field outbreaks of krimpsiekte, which usually occur during the winter to early summer. The highest cotyledoside concentrations were detected in the flowering stalk. Cotyledoside was not the only component of this type in the plant, as mass spectrometry revealed the presence of other, possibly related, compounds.

Nephrotoxicity of Narthecium ossifragum in cattle in Norway.

During the summer of 1992 renal failure was diagnosed in 232 grazing cattle in 85 herds on the west coast of Norway. The salient clinical signs were depression, anorexia and melaena or fresh blood in the faeces; diarrhoea was also commonly observed. The serum concentrations of creatinine, urea, magnesium and phosphorus, and the activities of glutamate dehydrogenase, aspartate aminotransferase and creatine kinase were above normal and the serum calcium concentration was below normal. Post mortem examinations consistently revealed renal tubular necrosis. In some cases there was liver necrosis and also erosions at the base of the tongue, in the oesophagus and in the jejunum and colon. The toxicity was probably caused by the plant Narthecium ossifragum (bog asphodel).

Parenchymal injury and biliary obstruction in relation to photosensitization in sporidesmin-intoxicated lambs.

The hepatic changes were compared in lambs photosensitized or not photosensitized after exposure to sporidesmin. Injury to both the parenchyma and the biliary system was more severe in the photosensitized than in the non-photosensitized lambs. The activities of gamma-glutamyltransferase and glutamate dehydrogenase, and the total, conjugated and unconjugated bilirubin concentrations were significantly higher in sera from the photosensitized than from the non-photosensitized lambs. Hepatic glycogen levels were decreased in both the photosensitized and the non-photosensitized lambs, but were significantly lower in the former. Hence it is possible that lesions in hepatocytes contribute to retention of phylloerythrin and so to photosensitization.

Microsomal enzymes in lambs and adult sheep, and their possible relationship to alveld.

The difference in susceptibility to alveld between lambs and adult sheep may be caused by differences in the microsomal enzyme activities in their livers. There was no difference in NADPH-cytochrome c reductase or 7-ethoxyresorufin-O-deethylase (EROD) activity between ewes, control lambs and phenobarbitone-dosed lambs 3 weeks after dosing ceased. However, aldrin epoxidase activity was at that time significantly highest in the phenobarbitone-dosed lambs and significantly lowest in the ewes. The liver cytosolic glutathione transferase activity was significantly highest in the ewes and significantly lowest in the control lambs at the same time.

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

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

Estimation of glomerular filtration rate in normal sheep by the disappearance of iohexol from serum.

The aim of the study was to establish reference values for glomerular filtration rate (GFR) in healthy sheep from the clearance of iohexol in serum. Fifteen healthy sheep were tested twice with 14 to 21 days between tests. No side-effects were observed after iohexol injections and all the sheep were clinically normal during and after the study. The mean clearance of iohexol estimated by the two-compartment method was 1.8 ml/min per kg (95% CI = 1.6-2.0) in the first trial and 1.7 ml/min per kg (1.5-1.9) in the second trial. The mean GFRiohexol estimated by a one-compartment method was 1.9 ml/min per kg (1.7-2.2) in the first trial and 1.8 ml/min per kg (1.6-2.0) in the second. The GFR values were similar to those reported for the inulin method. The results indicate that the iohexol method is valid for estimating GFR in sheep, and it is easier to perform than the inulin method.

Metabolism of saponins from Narthecium ossifragum--a plant implicated in the aetiology of alveld, a hepatogenous photosensitization of sheep.

One sheep was dosed over 4 consecutive days with 2.1 kg of leaves and flower stems of Narthecium ossifragum before it was killed. Sarsasapogenin and smilagenin glycosides, in the ratio 9:1, were the dominant saponins present in the dosed plant material. GC-MS analyses of the free and conjugated sapogenin content of samples recovered from the sheep identified three distinct regions of metabolic activity. In the first metabolic region, in the rumen and omasum, the ingested plant saponins were hydrolysed to the parent sapogenins, before being oxidized at C-3 and reduced to give the epi analogues of the ingested sapogenins. The second metabolic region consisted of the duodenum, jejunum, the liver and associated ducts. Sapogenins appear to be absorbed in the jejunum and may be transported via the portal vein to the liver, where 3 alpha-OH-5 beta-H sapogenins (epismilagenin and episarsasapogenin), but not 3 beta-OH-5 alpha-H sapogenins (smilagenin and sarsasapogenin), are conjugated and excreted into the bile as episarsasapogenin and epismilagenin conjugates in the ratio 4:1. In the third metabolic region, in the caecum and the colon, the epi-sapogenin conjugates were hydrolysed to free epi-sapogenins. The absence of free and/or conjugated sapogenins in urine, collected 24 h after dosing commenced, indicates that saponins and their metabolites are not likely to be implicated in the kidney disease occurring in ruminants ingesting N. ossifragum.

Nephrotoxicity in lambs apparently caused by experimental feeding with Narthecium ossifragum.

Seven lambs were fed a mixture of bog plants containing 15 g (wet matter) Narthecium ossifragum per kg live weight for 10 consecutive days. Their serum creatinine concentration increased from day 1 to 4 but then fell to normal by day 6 after feeding started. In the same animals, the serum magnesium concentration was increased on days 3, 4 and 5. Histopathological examination of the kidneys of 2 lambs killed after being fed 25 g N. ossifragum per kg live weight for one day revealed tubular epithelial cell degeneration and necrosis. There were no signs of disease or distress in the experimental animals and their appetite remained normal throughout the experimental period. It was concluded that N. ossifragum is probably nephrotoxic to lambs.

Aldrin epoxidase and 7-ethoxyresorufin-O-deethylase (EROD) activity in livers from lambs of three different breeds of Norwegian sheep.

The concentration of aldrin epoxidase was significantly greater in the livers of Norwegian Pelt lambs than in those of Rygja lambs but did not differ significantly between either of these two breeds and Spael lambs. Conversely, although there was no detectable EROD activity in the livers of many of the lambs in all three breeds, the median activity was greater in the livers of Rygja lambs than was the case for Spael lambs.

Tolerance to the nephrotoxic component of Narthecium ossifragum in sheep: the effects of repeated oral doses of plant extracts.

Young adult sheep were dosed with extracts of Narthecium ossifragum plants by the oral or parenteral routes and the resulting nephrotoxicity was assessed from the increases in the concentrations of creatinine and urea in the serum. Following single intraruminal or intraperitoneal doses of extracts derived from 30 g N. ossifragum (wet weight) per kg live weight (kg lw), serum creatinine concentrations increased from about 100 micromol/L to between 260 and 510 micromol/L. The serum urea concentrations increased from about 5-8 mmol/L to between 11 and 66 mmol/L in individual sheep. Daily intraruminal administration of 5-30 g/kg lw to three sheep over a 10- or 15-day period increased creatinine concentrations from 100 micromol/L to 300-760 micromol/L, and urea concentrations from 5-8 mmol/L to 35 mmol/L. A single intraperitoneal challenge dose of 30 g/kg lw, delivered 7 or 12 days after the final intraruminal dose, did not lead to increased serum creatinine or urea concentrations, indicating that oral treatment had apparently resulted in an increased tolerance to the nephrotoxic principle(s) in N. ossifragum.

Ruminal metabolism in sheep of saponins from Yucca schidigera.

Fifty-five mg per kg live weight of crystallized Yucca schidigera saponins, corresponding to 26 mg/kg live weight of sapogenins, was given daily intraruminally to two lambs for 11 consecutive days. Neither of the lambs showed any sign of toxicity throughout the experimental period. One lamb was killed 5 h after the last dose and GC-MS analysis of the free and conjugated sapogenin content samples of liver, and of the contents of the rumen, omasum, abomasum, duodenum, jejunum, ileum, colon and rectum, of faecal samples collected before dosing started, and of parts of the administered Yucca saponin were performed. The Yucca material contained mainly sarsasapogenin and smilagenin saponins. Ingested saponins were quickly hydrolysed in the rumen to free sapogenins and, in part, epimerized at C-3 to afford episapogenins. The absorption of free sapogenins appeared to occur in the jejunum. The metabolism of Yucca saponins was identical to that of Narthecium ossifragum saponins, and it is suggested that Yucca saponins could replace N. ossifragum saponins for toxicity studies on the latter plant.

Nephrotoxicity and hepatotoxicity in calves apparently caused by experimental feeding with Narthecium ossifragum.

Seven calves were fed a mixture of bog plants containing 15 g (wet matter) Narthecium ossifragum per kg live weight for two consecutive days. Their serum creatinine, urea and magnesium concentrations increased, whereas the serum calcium concentration decreased. Histopathological examination of the kidneys of the 5 calves that were killed revealed tubular epithelial cell degeneration and necrosis. There were signs of liver dysfunction in all the calves including increased aspartate aminotransferase (ASAT), glutamate dehydrogenase (GLDH) and gamma-glutamyltransferase activities. All the calves refused to ingest N. ossifragum after 2 days feeding, and their appetite for hay and concentrate was also reduced. It can be concluded that N. ossifragum is nephrotoxic and hepatotoxic to calves.

Failure to induce toxicity in lambs by administering saponins from Narthecium ossifragum.

It has been suggested that saponins produced by Narthecium ossifragum (Bog asphodel) may be the direct cause of the toxicity leading to the hepatogenous photosensitivity disease alveld seen in Norwegian lambs. Lambs fed large quantities of freeze-dried N. ossifragum did not develop alveld. Chemical investigations on the freeze-dried material and fresh N. ossifragum showed no difference in their saponin content. These results indicate that alveld is not caused solely by the saponins produced by N. ossifragum.

Fungi on Narthecium ossifragum leaves and their possible involvement in alveld disease of Norwegian lambs.

Spores of Pithomyces chartarum (Berk. & Curt.) M.B. Ellis were only rarely seen on leaves of Narthecium ossifragum (L.) Hudson collected in summer from five areas in western Norway in which alveld, a photosensitization disease of lambs, is endemic. Cladosporium magnusianum (Jaap) M.B. Ellis was found on all 118 leaf samples collected in the summers of 1990 and 1991. The hypothesis that P. chartarum contributes to the aetiology of alveld could not be supported, but it is possible that C. magnusianum may have a role in the causation of the disease.

Ovine metabolism of saponins: evaluation of a method for estimating the ovine uptake of steroidal saponins from Narthecium ossifragum.

A sheep was dosed three times per day over six consecutive days with 70 g Narthecium ossifragum, and once on the seventh day with 70 g N. ossifragum. Additionally, it was dosed once on days 1-7 with 20 mg of [20,23,23-2H3]sarsasapogenin. After 7 days, the sheep was killed and GC-MS analysis of the free and conjugated sapogenin content in bile, urine, rumen, duodenum, jejunum, colon and rectum samples collected from the sheep, faecal samples collected on days 4-7, and dosed plant material was performed. The N. ossifragum contained mainly sarsasapogenin and smilagenin. Only neglible levels of deuterium-labelled sarsasapogenins were detected in the samples from the animal. Ingested saponins were quickly hydrolysed in the rumen to free sapogenins and, in part, epimerized at C-3 to afford episapogenins. The absorption of free sapogenins appeared to occur in the jejunum. The concentration of sapogenins in faeces reached a plateau 108 h after dosing started.