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 alveld 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.Abbreviations HE haematoxylin and eoson - PAS periodic acid Schiff - EDTA ethylene diamine tetracetic acid     

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.     

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.     

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.     

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.     

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.     

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.     

Pathological changes in cobalt-supplemented and non-supplemented twin lambs in relation to blood concentrations of methylmalonic acid and homocysteine.

In a controlled field study of three years' duration we evaluated the effect of cobalt supplementation on pathological changes in cobalt/vitamin B12-deficient Texel twin lambs grazing the same cobalt-deficient pasture. Semi-quantitative evaluation of the histopathology of liver and brain was done on 44 sets of twins. Pathological changes were related to blood concentrations of vitamin B12, methylmalonic acid, and homocysteine. Lesions were mainly confined to the liver and brain. Acute hepatic changes were characterized by steatosis, hepatocytic degeneration, and single cell necrosis. Chronic changes consisted of bile duct proliferation, the presence of ceroid containing macrophages, and fibrosis in the portal triads. Many non-supplemented lambs showed polymicrocavitation and Alzheimer type II reaction in the brain. Polioencephalomalacia was observed in three non-supplemented lambs but was regarded as a secondary lesion. Our results indicate that the main lesions found in cobalt/vitamin B12-deficient lambs are acute and chronic hepatitis. These lesions were associated with low concentrations of vitamin B12 and high concentrations of methylmalonic acid and homocysteine in the blood. The liver lesions were also associated with polymicrocavitation of the brain, probably as morphological evidence of hepatoencephalopathy.     

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.     

Congenital polycystic kidney disease in lambs

AIM: To describe the pathology and inheritance of a congenital polycystic kidney disease (PKD) of sheep. METHODS: Mode of inheritance of PKD was investigated by evaluation of results of the disorder from planned matings in two consecutive years within subsets of a flock that had a high prevalence of PKD in lambs. Gross pathological and histopathological studies were based on tissues derived from this study. Haematoxylin and eosin (H&E)-stained paraffin sections of kidney, liver, extrahepatic biliary and pancreatic ducts, pancreas and epididymis were used to describe the lesions. RESULTS: Twenty-five lambs affected by PKD, of both sexes, were born, numbers in accord with those expected for an autosomal recessive disorder in the population studied. In all cases for which tissues were available, the renal, bile ductal (intrahepatic and extrahepatic), pancreatic and epididymal tissues had widespread dysplastic changes and associated cyst formation. CONCLUSIONS: The findings of renal cysts in conjunction with cysts in other organs are unifying features in many of the human and animal forms of PKD and suggest a related pathogenic and genetic base consistent with an autosomal recessive disorder.     

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.     

An outbreak of subacute fasciolosis in Soay sheep: ultrasonographic biochemical and histological studies

A naturally occurring outbreak of subacute fasciolosis in a group of 17 seven-month-old Soay ram lambs was studied following the sudden death of two sheep. In addition to standard biochemical investigations, ultrasound examination of the liver and cranial abdominal cavity was undertaken. There was a significant positive linear correlation between liver weight and ultrasonographic determination of liver size (R=0.72, P<0.05). Ultrasonographic examination of the liver revealed multiple hyperechoic dots in the parenchyma giving a granular appearance to the hepatic texture in three sheep corresponding to the most advanced histopathological changes as determined by the size of the abscesses and their relatively mature fibrous capsules, and areas of hepatic necrosis. No distension of the bile duct system was noted nor was the gall bladder imaged. While serum concentrations of albumin, globulin and certain liver enzymes assisted in the diagnosis of subacute fasciolosis in sheep, only glutamate dehydrogenase, and gammaglutamyl transferase remained elevated four weeks after triclabendazole treatment.     

Ovine dermatophilosis in young sheep associated with the grazing of Brassica spp. crops

The occurrence of dermatophilosis in five month old lambs associated with the grazing of Brassica spp. crops is reported on four farms. The prevalence of the disease varied from 3% to 45% within affected flocks. An additional finding was the occurrence of nervous signs in a small number of more severely affected lambs, characterised by a sudden lowering of the hindquarters.

The association between dermatophilosis and the grazing of Brassica spp. crops is discussed.     

Retention of selenium in tissues of calves, lambs, and pigs after parenteral injection of a selenium-vitamin E preparation.

Four each healthy weaned calves, lambs, and pigs raised in Indiana without selenium supplementation were killed, and their tissues were fluorometrically analyzed to establish base line selenium concentrations. The following mean selenium content (in ppm, wet weight) was found in calves, lambs, and pigs, respectively: liver, 0.12, 0.16, and 0.19; renal cortex, 0.63, 0.89, and 0.70; muscle, 0.05, 0.05, and 0.06. Eight each additional healthy weaned calves, lambs, and pigs were injected with a commercial selenium-vitamin E preparation at dose levels of 0.0825, 0.055, or 0.06 mg of Se (as selenite) per kilogram of body weight, respectively. Selenium content of tissues was measured in animals killed at 1, 7, 14, and 23 days after injection. In calves, concentrations in liver and kidney rapidly increased to moderate values and then slowly decreased, with mean concentrations after 23 days still somewhat greater than base line values. Concentrations for injection site tissue also rapidly increased to moderate values, but had decreased to base line values by 23 days after injection. In lambs, selenium content of liver was moderately increased after injection, but had decreased to base line values after 14 days; kidney and injection site did not have increased selenium content after injection. In pigs, liver and kidney had moderate initial increases in concentration of selenium, but these were at base line values after 14 days, and increase did not occur at injection sites.     

Suspected photosensitisation in lambs grazing birdsfoot trefoil (Lotus corniculatus)

Suspected photosensitisation occurred in three groups of lambs grazing birdsfoot trefoil (Lotus corniculatus c.v. Grasslands Goldie). In one group, sucking lambs aged about 10 weeks, grazing birdsfoot trefoil, developed skin lesions while lambs of a similar age and from the same flock grazing lucerne (Medicago sativa) or a mixed sward of both species showed no signs of photosensitisation. Affected lambs had lesions on their backs and ears. In a few animals the tips of the ears were shortened by 2-3 cm. In the affected lambs, serum liver enzymes (gamma-glutamyltransferase, glutamate dehydrogenase), bilirubin and serum Vitamin B12 levels were within the normal range. At necropsy, no significant pathological changes were detected in the liver and histological changes in the skin were consistent with primary photosensitisation. In the second group, three of 80 weaned lambs grazing the same birdsfoot trefoil at a restricted intake were affected in the same manner as the first group. In the third group, 15 animals from 28 sets of sucking twin lambs were also affected. In only two sets of twins were both lambs affected. None of the ewes grazing with the lambs in the first or third groups showed any clinical signs of photosensitisation.     

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.     

A study of high lamb liver copper concentrations on some farms in Otago and Southland

Lamb copper status is commonly assessed by measuring copper concentrations in four liver samples collected from lines of lambs sent to meat slaughtering premises. High liver copper concentrations were found in lambs examined in April and May. Five farms with two or more lamb liver copper concentrations greater than 3000 micromol/kg on a wet matter basis and two farms with adequate copper concentrations but less than 3000 micromol/kg in the autumn of 1992 were selected for a more detailed investigation into the factors affecting their lamb copper status in 1993. High liver copper status was associated with low pasture molybdenum, grazing paddocks recently topdressed with copper sulphate, supplementing with mineralised drenches and copperised salt licks, and the high copper content in chicory. Lamb liver copper concentrations were significantly (p<0.05) higher in the autumn than in the summer on two of three farms.     

A study of high lamb liver copper concentrations on some farms in Otago and Southland

Lamb copper status is commonly assessed by measuring copper concentrations in four liver samples collected from lines of lambs sent to meat slaughtering premises. High liver copper concentrations were found in lambs examined in April and May. Five farms with two or more lamb liver copper concentrations greater than 3000 𝛍mol/kg on a wet matter basis and two farms with adequate copper concentrations but less than 3000 𝛍mollkg in the autumn of 1992 were selected for a more detailed investigation into the factors affecting their lamb copper status in 1993. High liver copper status was associated with low pasture molybdenum, grazing paddocks recently topdressed with copper sulphate, supplementing with mineralised drenches and copperised salt licks, and the high copper content in chicory. Lamb liver copper concentrations were significantly (pp<0.05) higher in the autumn than in the summer on two of three farms.     

The effect of increasing the Vitamin B12 status of Romney ewes on foetal liver Vitamin B12, milk Vitamin B12 and liver Vitamin B12 concentrations in suckling lambs

AIM: To determine the effect of increasing the Vitamin B12 status of the ewe on the Vitamin B12 supply to the suckling lamb. METHODS: The Vitamin B12 status of the ewe was increased during gestation and lactation by three injections of a long-acting preparation of Vitamin B12 microencapsulated in an organic acid polymer. The Vitamin B12 status of the ewes and suckling lambs was assessed from changes in serum and liver Vitamin B12 concentrations. RESULTS: Compared to untreated animals, serum and liver Vitamin B12 concentrations of the treated ewes were increased at least 70% during gestation. Foetal liver Vitamin B12 concentrations were increased 270%. Over the lactation, ewe serum and milk Vitamin B12 concentrations were increased at least 200% and 44%, respectively. The liver Vitamin B12 stores of the newborn lambs from Vitamin B12-treated ewes were depleted within 58 days. There were no significant differences in the serum Vitamin B12 concentrations of suckling lambs from Vitamin B12-treated and untreated ewes. CONCLUSION: Ewes with a high Vitamin B12 status will ensure an adequate supply of Vitamin B12 to their lambs for at least the first 30 days of life. CLINICAL SIGNIFICANCE: In flocks grazing Co-deficient pastures, treating ewes with a long-acting Vitamin B12 supplement at mating will prevent Vitamin B12 (Co) deficiency in ewes, as well as their lambs, until they can be treated at tailing at 4-6 weeks of age.     

Residual effects of Mycobacterium avium infection on susceptibility of sheep to copper toxicity and efficacy of treatment with tetrathiomolybdate

Posthaemolytic copper poisoning (post-HCP) in one of six, one-year-old, uninfected sheep (group O) on a Mycobacterium avium experiment prompted an evaluation of copper status and hepatotoxicity in 17 surviving cohorts. Group O had higher mean plasma Cu and δ-glutamyl transferase (GGT) activity and more variable bile acid (BA) concentrations and glutamate dehydrogenase activities (GDH) than two groups infected with M avium soon after birth. Ammonium tetrathiomolybdate (TTM; 3 x 1.7 mg/kg LW) was given subcutaneously over seven days and the pelleted, complete diet replaced by hay, low in copper. Plasma BA immediately declined and was followed by GDH, but erythrocyte superoxide dismutase activity (ESOD) became severely inhibited and took 18 days to recover. Plasma BA and GDH rose sharply after 18 days in uninfected sheep and they became hypercupraemic. TTM treatment was repeated from day 42 and had removed all group differences by day 110 but only after further inhibition of ESOD. M avium infections probably lessened the severity of pre-HCP by reducing copper retention but may predispose grazing livestock to hypocupraemia. The capacity of TTM to reduce liver Cu has probably been overestimated and side effects on cuproenzyme activity underestimated.