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.

Summer variation in the concentration of steroidal sapogenins in and the degree of fungal infection on Narthecium ossifragum plants from Møre og Romsdal County, Norway.

Thirty-nine leaf samples of Narthecium ossifragum collected from eight sites in Møre og Romsdal County, Norway, during June-September 1997 and 41 leaf samples collected at five sites in the same county during June-August 1998 were analysed for the concentrations of steroidal sapogenins using GC-MS. The 1998 samples were also examined for fungal elements (conidia and hyphae) after incubation in a moist chamber for 10-14 days. The highest 1997 and 1998 leaf sapogenin concentrations (4881 and 7115 mg/kg dry matter, respectively) were 13-14 times greater than the lowest sapogenin concentrations found (344 and 531 mg/kg dry matter, respectively). The results did not reveal systematic differences in sapogenin concentrations between the two seasons, or between samples harvested early or late in the same seasons, or between sapogenin concentrations in plants harvested at different sites. Cladosporium magnusianum was the predominant fungus found in the samples. The degree of fungal infection on the samples was in generally low, but the number of C. magnusianum colonies in the moist chamber preparations and fungal elements (conidia and hyphae) in leaf washings and on leaves tended to increase with time. Factor analysis and multiple regression analysis performed on the chemical and fungal results suggest that sporulation may have occurred in the fungi in response to increase in sapogenin concentrations.

Toxicokinetics of cotyledoside following intravenous administration to sheep.

Cotyledoside, a bufadienolide cardiac glycoside, was administered intravenously to sheep in 2 studies. In experiment 1, sheep (n = 4) received 0.0135 mg/kg daily on 5 consecutive days and in the 2nd experiment, sheep (n = 4) received 0.027 mg/kg as a single dose. Jugular blood was collected at different time intervals and kinetic parameters were determined. The data fitted a 1-compartmental model. In both experiments a short half-life (t1/2) and mean residence time (MRT), a relative small volume of distribution (Vd(ss)) and rapid clearance were calculated. In the 1st experiment, t1/2 and MRT increased significantly (P < 0.007) from Day (D) 0 to D4. It is suggested that the rapid decline in plasma cotyledoside concentrations in sheep denotes rapid distribution of cotyledoside to the tissues or extracellular spaces and possible accumulation at the biophase.

Narthecium ossifragum (L.) huds. causes kidney damage in goats: morphologic and functional effects.

We studied the effects of Narthecium ossifragum on goat kidneys. Twenty-five Norwegian dairy goats, 5 weeks to 4 months of age, were orally dosed with an aqueous extract from N. ossifragum. In experiment 1, we studied microscopic and functional changes in 12 animals that were euthanatized 2, 3, 4, 5, and 6 days after treatment. In experiment 2, we included ultrastructural studies on serial renal biopsies and urine analysis from five extract-treated animals and two controls. In addition, urine samples were collected from four dosed and two control goats. Ultrasonography revealed perirenal and retroperitoneal fluids. Microscopic changes were observed after 6 hours. The findings, most obvious in the inner cortex and the outer medulla, consisted of cytoplasmic vacuolization, interstitial edema, and focal necrosis of tubular epithelial cells. Ultrastructurally, the tubules had loss of microvilli, irregular cytoplasmic vacuolization, mitochondrial swelling with loss of cristae, and irregular but continuous basement membranes even with necrosis. In the glomeruli, there were occasional endothelial damage and shortening and swelling of the foot processes. Peritubular capillaries had breaks in the vessel walls and irregular endothelial cell edema, and the interstitium had marked edema. The functional lesions included elevated serum urea, creatinine, and magnesium concentrations, a slight decrease in serum calcium concentration, elevated urine protein and urine protein-creatinine ratio, and increased activities of urine alkaline phosphatase and gamma glutamyl transferase. Our findings indicate a fast-acting toxic principle inducing damage by both direct toxic and secondary ischemic effects.

Measurement of phylloerythrin (phytoporphyrin) in plasma or serum and skin from sheep photosensitised after ingestion of Narthecium ossifragum.

AIM: To establish a method for measuring phylloerythrin in plasma or serum and skin from lambs photosensitised after ingestion of the plant, Narthecium ossifragum, which induces an hepatogenous photosensitisation similar to the disease known as facial eczema in sheep. METHODS: For two successive summers, lambs were grazed on uncultivated pastures containing N. ossifragum. Clinical photosensitisation was deemed to have occurred when symptoms such as restlessness, scratching, oedema and reddening of the skin were observed. Sixteen lambs that exhibited signs of photosensitisation were included in this study in the first year and five in the following year. A total of 16 clinically healthy lambs served as controls. Fluorescence emission and excitation spectra of phylloerythrin were measured in plasma or serum samples from the 21 photosensitised and 16 non-photosensitised lambs. In the first year of the study, skin samples were collected post mortem from the ear, lip, neck, nose, leg, belly, udder, back, vulva and perineal region, from all photosensitised and from seven non-photosensitised lambs, and examined by fixing them between two glass plates (each of 1 mm thickness) and placing them at a fixed angle in front of a fluorescence spectrofluorometer. RESULTS: All plasma or serum samples obtained from the photosensitised lambs exhibited strong phylloerythrin-like fluorescence of identical spectra; maximum fluorescence was at 650 and 711 nm, and maximum excitation at 425 nm. Emission spectra obtained from plasma or sera from non-photosensitised sheep grazing the same N. ossifragum-containing pastures exhibited either no or only minor fluorescence. Phylloerythrin concentration in plasma or serum exceeded 0.3 microg/ml before clinical photosensitisation occurred, whereas the concentration in samples from clinically healthy lambs was <0.05 microg/ml. Fluorescence from skin samples from the photosensitised lambs showed emission peaks at 650, 670 and 711 nm, whereas the phylloerythrin emission peaks at 650 and 711 nm were not observed in skin from clinically healthy lambs. CONCLUSION: Plasma concentrations of phylloerythrin in healthy sheep were <0.05 microg/ml. Clinical signs of photosensitisation were not observed until the concentration of phylloerythrin in plasma exceeded 0.3 microg/ml. This is the first reported spectroscopic method for analysis of phylloerythrin and the only one which does not involve exposure of the analyst to hazardous chemicals. It has the additional benefit of distinguishing between hepatogenous and primary photosensitisation.

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.

Fluorescence spectra and measurement of phylloerythrin (phytoporphyrin) in plasma from clinically healthy sheep, goats, cattle and horses.

AIM: To measure the background concentration of phylloerythrin in plasma from clinically healthy sheep, goats, cattle and horses on pasture. METHODS: Blood samples were taken from 34 sheep of the Dala breed, 20 female Norwegian dairy goats, 35 Norwegian Red cows and 34 horses of different breeds. All animals were grazing green pasture when blood samples were taken. Blood samples were collected from each of four clinically healthy newborn lambs, goats, calves and foals, and pooled into one sample per species. Plasma samples were analysed for phylloerythrin by fluorescence spectroscopy, using a Perkin-Elmer LS-50B luminescence spectrometer equipped with a red-sensitive photomultiplier. The fluorescence spectra of phylloerythrin in plasma from the adult ruminants were compared with those in plasma from the neonatal ruminants, to which a known concentration of phylloerythrin had been added. RESULTS: Plasma obtained from the adult ruminants had spectral characteristics similar to those of phylloerythrin, namely weak emission peaks at 650 and 711 nm, when excited at 425 nm. Emission spectra obtained from plasma from the neonatal ruminants showed no fluorescence at these wavelengths. On average, 0.012 (SD 0.004), 0.06 (SD 0.04), and 0.05 (SD 0.03) micromol/l phylloerythrin were present in plasma samples from the sheep, goats, and cattle, respectively. The fluorescence spectra of plasma from the newborn foals were similar to spectra of plasma from adult horses, with weak emission at 669 nm. CONCLUSION: Small concentrations of phylloerythrin were detected in plasma from clinically healthy sheep, goats and cattle, but none could be detected in plasma from clinically healthy horses.

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.

Accumulation of sapogenin conjugates and histological changes in the liver and kidneys of lambs suffering from alveld, a hepatogenous photosensitization disease of sheep grazing Narthecium ossifragum.

Sixteen lambs exhibiting hepatogenous photosensitization (alveld) after grazing pasture containing Narthecium ossifragum and seven nonphotosensitized lambs grazing the same pastures were studied. All the alveld-affected lambs revealed liver damage dominated by single cell necrosis, portal fibroplasia and bile duct proliferation. Crystalloid clefts were demonstrated in the bile ducts of two and in the hepatocytes and Kupffer cells of nine photosensitized lambs. Plasma bilirubin concentration was severely increased in ten of the cases of alveld whereas the activity of aspartate aminotransferase was moderately to severely increased in seven cases. The activity of glutamate dehydrogenase was moderately elevated in one of the photosensitized lambs. The main histopathological findings in the kidneys from the alveld-affected lambs were dilated tubules, often with eosinophilic material in the tubular lumina. Regenerative changes were seen in a large proportion of the renal sections. Elevated plasma concentrations of urea and creatinine, and the renal histopathological changes, suggested that the photosensitized lambs had been through a phase of renal injury. Analysis of the free and conjugated sapogenin content in liver tissue and bile was performed by gas chromatography-mass spectrometry. There were significantly higher concentrations of conjugated episapogenins in both the liver and bile in the alveld-affected lambs than in the nonphotosensitized lambs.

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.

Phylloerythrin. mechanisms for cellular uptake and location, photosensitisation and spectroscopic evaluation.

AIM: To elucidate the photobiological behaviour of phylloerythrin by studying the cellular uptake and intracellular localisation pattern of phylloerythrin and its spectral properties in Chinese hamster lung fibroblast cells (V79). METHODS: Phylloerythrin was diluted in dimethylsulfoxide (DMSO). Fluorescence emission and excitation spectra were measured using a luminescence spectrometer equipped with a red-sensitive photomultiplier. V79 cells were cultured in monolayers and labelled with 0.25 microg/ml phylloerythrin for uptake, cell survival and intracellular localisation studies. For cell survival and intracellular localisation studies, cells were subsequently exposed to blue light at a fluence rate of 9.0 mW/cm2. RESULTS: The fluorescence excitation spectrum of phylloerythrin in DMSO was characterised by a Soret band exhibiting a maximum peak at 418 nm. The fluorescence emission spectrum had peaks at 643 and 706 nm. The corresponding spectra in cells were red-shifted to 422, 650 and 712 nm, respectively. The cellular uptake of phylloerythrin was complete after about 10 h of incubation. The uptake together with the activation energy and analysis of cells incubated with phylloerythrin at 37 degrees C and 0 degrees C using fluorescence microscopy indicated that the dye is taken up into cells via a diffusion mediated pathway. Measurements of subcellular marker enzymes were performed immediately after light exposure of phylloerythrin-treated cells. The mitochondrial marker enzyme, cytochrome-c oxidase, and the marker enzyme for the Golgi apparatus, UDP galactosyl transferase, but not those for lysosomes, -N-acetyl-D-glucosaminidase (-AGA), and endoplasmic reticulum, NADPH cytochrome-c reductase, were inactivated upon photodynamic treatment. CONCLUSION: These results indicate that phylloerythrin is located mainly in the Golgi apparatus and mitochondria of V79 fibroblasts cells.

Tremorgenic neuromycotoxicosis in 2 dogs ascribed to the ingestion of penitrem A and possibly roquefortine in rice contaminated with Penicillium crustosum.

Two dogs developed alarming tremorgenic nervous stimulation shortly after ingesting discarded rice that had been forgotten in a refrigerator for an undetermined period and that was covered with a grey-green mould. Both dogs exhibited vomition followed by slight salivation, tremors and ataxia and 1 showed such severe agitation and seizures that it necessitated anaesthesia with thiopentone followed, on recovery, by xylazine. The other dog was only sedated with xylazine. They made an uneventful recovery. The rice vomitus yielded a pure culture of Penicillium crustosum. On chemical analysis it was negative for organochlorine, organophosphor and carbamate insecticides, as well as for strychnine, but contained 2.6 microg/g of the mycotoxins penitrem A as well as 34 microg/g of roquefortine as determined by LC-MS and confirmed by MS-MS. This is the 1st South African case of naturally occurring penitrem A toxicosis and also the 1st case where quantification of the levels of mycotoxins in dog vomitus is reported. The tremorgenicity of roquefortine and its contribution towards this syndrome, is questioned.

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.

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.

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.

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.

The possible involvement of 3-methoxy-2(5H)-furanone in the etiology of Narthecium asiaticum maxim. associated nephrotoxicity in cattle.

Two samples of Narthecium asiaticum Maxim leaves collected in Japan were found to contain 103 microg(-1) and 160 microg g(-1) dry matter of 3-methoxy-2(5H)-furanone respectively. 3-Methoxy-2(5h)-furanone was suggested to be the toxic principle of N. asiaticum causing nephrotoxicity in cattle in Japan. Two other furanones, which are thought to be non-toxic, were also isolated from the two samples. These were 4-methoxy-2(5H)-furanone and 5-hydroxy-4-methoxy-2(5H)-furanone.