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.     

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.     

Alveld-Producing Saponins: II. Toxicological Studies

The phototoxic lamb disease alveld, prevalent in South-Western Norway, is caused by ingestion of Narthecium ossifragum. Earlier studies have shown that peroral administration of large amounts of crude saponins from this plant elicits the disease. Such saponins have now been purified further by 2 different methods (A and B). Two A type preparations resulted in alveld when fed to 2 lambs. The most highly purified preparation (type B) did not cause alveld in the 2 lambs tested. Lambs vary, however, in their susceptibility to the disease. Both types of preparations led to increases in serum aspartate aminotransferase, bilirubin and 5′-nucleotidase in rats when injected intraperitoneally in amounts of 50 or 100 mg/kg body Weight. Cannulation of the bile duct showed that injected saponins reduced both the volume of bile and the amounts of bilirubin and bile acids excreted. Histological changes seen in the light microscope were, except for the most peripheral parts of the liver, hardly noticable. These observations support the view that saponins are the liver-toxic agents responsible for alveld. The possibility is discussed that the effect arises through a change in the lipid environment of carrier-mediated transport systems.     

Detection of singlet oxygen in blood serum samples of clinically healthy lambs and lambs suffering from alveld disease

Alveld is a disease in lambs of domestic sheep (Ovis aries L.), characterized by a combination of photosensitivity and liver damage. Generation of singlet oxygen play a major role in phototoxicity reactions. The compound phylloerythrin (phytoporphyrin) is so far assumed to be the main photodynamic agent in hepatogenous photosensitivity diseases in sheep. Phylloerythrin is a potent photosensitizer and an efficient source of singlet oxygen. The compound accumulates in the peripheral circualtion upon liver damage. Liver dysfunction is also likely to cause an increase in the blood level of bilirubin. Formation of singlet oxygen by bilirubin is reported. In the present work the photosensitizing potential of serum has been measured and related to the bilirubin- and phylloerythrin levels in lambs suffering from alveld and in clinically healthy controls. The singlet oxygen level of the serum was taken as a measure of the photosensitizing potential. The observed singlet oxygen values in serum from alveld lambs were significantly higher than the corresponding values observed in clinically healthy control lambs. This indicates that the serum of the alveld lambs contains an elevated concentration of photosensitizer. The singlet oxygen level was not correlated to the concentration of bilirubin or phylloerythrin. The results indicate that the photosensitizing mechanism is quite complex and may involve other sensitizer(s) than phylloerythrin.     

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 -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.     

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 onNarthecium ossifragum leaves and their possible involvement in alveld disease of Norwegian lambs

Spores ofPithomyces chartarum (Berk. & Curt.) M.B. Ellis were only rarely seen on leaves ofNarthecium 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 thatP. chartarum contributes to the aetiology of alveld could not be supported, but it is possible thatC. magnusianum may have a role in the causation of the disease.     

Sapogenin Levels in <Emphasis Type="Italic">Narthecium ossifragum</Emphasis> Plants and <Emphasis Type="Italic">Ovis aries</Emphasis> 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.     

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.     

Ovine white-liver disease (OWLD). Pathology

Microscopic liver changes could earliest be found after 1 month on OWLD pasture, and include extensive fatty change with large spherical vacuoles in hepatocytes, varying size of hepatocytes and nuclei, and formation of Councilman bodies. Later came ceroid deposits, biliary hyperplasia and mesenchymal proliferation. Changes occurred in all lambs which died or were killed due to OWLD, and altogether 83% of the lambs grazing OWLD pastures showed typical or suspect changes. Widespread haemosiderosis of the spleen was common. In severely affected lambs, sclerosis of the Peyer's patches and of the germinative centres of the intestinal lymph nodes were seen, as were neuronal atrophy and patchy microcavitation of areas in the brain stem. Four had polyvasculitis. Cobalt/vitamin B12 supplemented lambs showed no specific changes. Lambs which grew well on other pastures (H lambs), but which were subclinically Co/B12 deficient some years, showed no fulminant hepatic OWLD, but 15% developed some features seen in OWLD. They showed no extensive fatty change. Results indicate that OWLD is a manifestation of B12 deficiency worsened by factors triggering early hepatic fatty change resulting in a more severe liver damage with loss of intracellular homeostasis rendering the hepatocytes vulnerable to other elements, like copper.     

Characterization of sublethal microcystin-LR exposure in mice

Microcystin-LR (MCLR) is a potent hepatotoxin produced by the cyanobacterium Microcystis aeruginosa. The histology of acute lethal toxicity has been well characterized, but histology is limited regarding sublethal exposure. Balb/C mice were given a single sublethal dose of MCLR (45 microg/kg) and euthanized at 2, 4, 12, and 24 hours after exposure. Centrilobular to midzonal hepatocellular hypertrophy with loss of cytosolic vacuolation consistent with glycogen depletion occurred at 2 hours. At 4 hours, central lobular hepatocytes exhibited eccentric areas of eosinophilic cytoplasmic condensation that were partially aggregated around the outer nuclear membrane. The areas were weakly positive for cytokeratin and somewhat resembled the Mallory bodies of alcoholic human hepatitis. Small numbers of apoptotic hepatocytes were seen at 24 hours. The toxin was detectable by immunohistochemistry (IHC) as early as 2 hours and was colocalized with the areas of hepatocellular hypertrophy. Intense nuclear staining occurred at 4 hours; this was no longer evident after 12 hours. Strong staining of apoptotic bodies occurred at 24 hours. Mice that received two daily doses had a marked increase in apoptotic hepatocytes in the centrilobular areas. Lesions at four and seven doses consisted of marked hepatocytomegaly and karyomegaly with parenchymal disarray and cytosolic vacuolation. IHC revealed diffuse staining throughout the liver parenchyma consistent with toxin accumulation. An anti-MCLR monoclonal antibody detected bands at the 40-kDa mark in nuclear extracts that were identified as protein phosphatases 1 and 2A by western blotting, consistent with a covalent interaction between MCLR and nuclear protein phosphatases.     

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.     

Field cases of aflatoxicosis in pigs

SUMMARY Five cases of aflatoxicosis in pigs in southern Queensland are described. One peracute case where aflatoxin concentrations of up to 5000μg aflatoxin B₁/kg were demonstrated in stomach contents was presumed to be caused by consumption of mouldy bread. High levels of toxins were also present in the livers. Two cases of acute toxicity were caused by feeding mouldy peanut screenings containing 22000μg aflatoxin B₁/kg. One case of subacute and one of chronic toxicity were caused by sorghum grain based rations with lower aflatoxin levels (4640 and 255 μg/kg). Peracute toxicity caused collapse and deaths within several hours, acute toxicity caused deaths within 12 h and with subacute toxicity deaths occured after 3 weeks on a toxic ration. Anorexia and ill thrift affecting only growing animals were seen with chronic toxicity. Extensive centrilobular liver necrosis and haemorrhage occured with peracute toxicity and in cases of acute poisoning there was hepatic centrilobular cellular infiltration, hepatocyte swelling and bile stasis. With subacute toxicity hepatocyte vacuolation together with bile stasis and bile ductule hyperplasia were seen.     

Acute aflatoxicosis in swine: clinical pathology, histopathology, and electron microscopy

Feeder pigs weighing 12 to 15 kg each were given a single oral dose of aflatoxin, 1.2 mg/kg of body weight. Liver-specific serum enzyme activities were compared with gross, microscopic, and ultrastructural hepatic changes in individual pigs euthanatized at 24, 48, and 72 hours after they were given aflatoxin. The greater the morphologic change in liver of the treated pigs, the greater the increase in liver-specific serum enzyme activities. Isocitric dehydrogenase, alkaline phosphatase, sorbitol dehydrogenase, and aspartate aminotransferase activities increased in 6 of 8 treated pigs by 24 hours. Increase in gamma-glutamyl transpeptidase activity was not significant. Microscopic and ultrastructural changes in centrilobular hepatocytes included glycogen deletion, mitochondrial and endoplasmic reticulum swelling, membrane disruption, and nuclear fragmentation at 24 hours. The centrilobular areas had marked extravasation of erythrocytes at 24 hours without basal lamina changes. At 72 hours, the centrilobular hepatocytes had increased lipid vacuoles and acceptable amounts of glycogen. Marked infiltrations of monocytes, plasma cells, and lymphocytes were also present at this time.     

Crystal-associated cholangiohepatopathy and photosensitisation in lambs

Four outbreaks of hepatogenous photosensitisation occurred in weaned lambs in north eastern Victoria during the summers of 1985 and 1986. Attack rates varied between 7% and 43% and case fatality rates between 60% and 71%. Clinical signs included photosensitisation and jaundice. Serum biochemistry suggested hepatobiliary and hepatoparenchymal damage with impaired renal function. At necropsy livers were an ochre colour and kidneys a mottled brown to khaki. Histopathologically, needle-shaped to lenticular clefts were observed in and around bile ducts and in hepatocytes, hepatic sinusoidal macrophages and renal tubules. Optically active rhomboidal crystals were present in bile sediments. Panicum schinzii was identified as a possible cause in 2 of the 4 outbreaks. The clinical disease was reproduced in 2 of 6 lambs grazed on a toxic paddock. The disease was indistinguishable from geeldikkop except for the fact that Tribulus terrestris was not present on any of the 4 farms.     

Changes in the concentrations of bile acids in the plasma of sheep with liver damage

The concentration of total plasma bile acids was measured in normal sheep and in sheep in which liver damage was induced by chronic copper poisoning, ligated bile ducts or induced ketosis. All three treatments produced a rise in total bile acid concentration in plasma which was proportional to the degree of hepatic damage seen histologically and which tended to parallel changes in activity of iditol, and glutamate dehydrogenase and aspartate amino-transferase in plasma. Plasma bile acid concentration was a more sensitive method of detecting these types of liver damage than was the measurement of total plasma bilirubin concentration, and could be used to assess alterations in liver function in sheep.     

Studies on the pathology of dicrocoeliasis and fascioliasis in the goat. I. The histopathology of the liver and bile ducts

Comparative studies were made on the histopathology of the liver and bile ducts in dicrocoeliasis and fascioliasis of the goat. Investigations revealed certain similarities in the nature of the pathological phenomena produced by D. dendriticum and F. hepatica. However, a feature peculiar to fascioliasis was the migratory tracks of immature flukes in the liver parenchyma. Further, the lesions produced by fascioliasis were distinctly more pronounced than those present in dicrocoeliasis.In both parasitic diseases the bile-duct walls showed glandular hyperplasia, increased frequency of goblet cells and globule leucocytes, and extensive fibrosis in the outer layers. The desquamation produced by Dicrocoelium remained superficial whereas Fasciola was capable of causing deep erosions in the walls of the main bile ducts. The bile-duct walls did not become calcified. Increased fibrosis was present in the interlobular areas, too, but only fascioliasis produced extensive disorganisation of the hepatic architecture, due to the scars originating from repaired migratory tracks. The liver parenchymal cells were degenerated, showed decreased glycogen but were increasingly infiltrated by neutral fats.Keyword: Dicrocoelium dendriticum, Fasciola hepatica, liver, bile ducts     

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.     

Quantitative Histomorphology of Liver Growth in Sheep at Prenatal and Postnatal Stages

This study was carried to determine quantitative histomorphologically on the development of the liver of sheep in prenatal and postnatal stages, and to prove the relationship between functional and structural differentiation of liver. There were more blood cells than hepatocytes, and haemotopoieisis was the primary function of the liver in the first half of gestation. As observed in the fetal stages bile ducts and Kiernan areas are formed from the 12th week. The distance between the two adjacent central veins was 401.2 ? 20.8 micron in the fetuses and 629.77 ? 34.7 micron in the lambs, while rising in the adult to 740 + 14.35 micron. This increase was directly proportional to age. The average diameter of sheep hepatocyte and nuclei, and the ratio between the diameters of nuclei and their hepatic cells were compared according to the prenatal and postnatal stages, and the difference between these stages was found to be statistically significant (P < 0.05). This difference was sourced from adult sheep. The number of hepatocytes per unit area were 107.48 ? 6.63, 133.6 ? 7.01, 100.84 ? 6.63 in the fetus, lamb and adult liver of sheep, respectively, and the differentia that earned statistical importance was sourced from the young stages. The number of ductus biliferi was 2.75 ? 0.47 in the fetuses, however, this had risen to 5.8 ? 0.6 and 6.8 ? 0.37 respectively in the lambs and adult sheep. The portai lobule areas rose according to the age and were 0.17796 ? 0.00086 mm² and 2.022650 ? 0.0097 mm² respectively in the lambs and adult sheep and the differentia between young and adult sheep was statistically important (P < 0.05).