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.     

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 photo- multiplier. 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) μmol/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.     

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.     

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.