Veterinay Research Communications, 15 (1991) 443-453 Copyright
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GLYCOGEN ACCUMULATION AND HISTOLOGICAL IN THE LIVERS OF LAMBS WITH ALVELD AND EXPERIMENTAL SPORIDESMIN INTOXICATION A. FL.&P)YEN’, ‘Department of ‘Department of Dep., 0033 Oslo
CHANGES
B. BORREB/EK2 AND K. NORDSTOGA3 Large Animal Clinical Sciences, 2Department of Biochemistry, and Pathology, Norwegian College of Veterinary Medicine, POB 8146 1, Norway
ABSTRACT Plgeyen, A., Borrebzk, B. and Nordstoga, K., 1991. Glycogen accumulation and histological changes in the livers of lambs with alveld and experimental sporidesmin intoxication. Veterina y Research Communications, 15 (6), 443-453 Alveld is a hepatogenous photosensitization disease seen in lambs grazing Narthecium ossijkagum 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-&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.
Keywords: alveld,
bog asphodel,
facial
eczema,
glycogen,
liver,
panicum,
photosensitization,
sheep,
sporidesmin
INTRODUCTION Both alveld and facial eczema are known to cause hepatogenous photosensitivity diseases of sheep. Facial eczema is most commonly seen in New Zealand, being caused by the toxin sporidesmin, which is produced by the saprophytic microfungus Pithomyces churtarunt (Mortimer, 1%3). Alveld is seen in lambs grazing pastures containing Nurthecium ossifragum (Bog asphodel) on the west coast of Norway (Laksesvela and Dishington, 1983). It has been suggested that saponins produced by N. ossijkgum cause the liver damage, which leads to retention of phylloerythrin (Abdelkader et al., 1984). However, Flaayen (1991) was unable to detect liver damage in lambs fed N. ossifugum containing saponins. Aas and Ulvund (1989) suggested that mycotoxins, possibly sporidesmin, might be involved in the pathogenesis of alveld. ABBREVIATIONS HE: haematoxylin
and eoson;
PAS: periodic
acid Schiff;
EDTA:
ethylene
diamine
tetracetic
acid
444
The present work was undertaken to study the hepatic lesions associated with alveld and to compare the histological changes in the liver of alveld and sporidesminintoxicated cases to see whether the histological findings indicate a similar pathogenesis.
MATERIALS
AND
METHODS
Liver tissue was collected from six White Norwegian lambs of both sexes, 2-2.5 months old, with clinical signs of photosensitization. The lambs followed their mothers grazing on uncultivated fields, mainly bogs containing large quantities of Narthecium ossifragum. The sheep were inspected approximately once a week. Lambs with clinical signs were killed with intrajugular injections of pentobarbitone. Liver tissue was also collected from a further eight White Norwegian lambs of both sexes, 1.5-2 months old, all of which had developed clinical signs of photosensitization after being dosed with 0.5 mg sporidesmin/kg body weight. The sporidesmin, obtained from Ruakura Agricultural Centre, Hamilton, New Zealand, was dissolved in 96% ethanol, diluted in water to 10% ethanol and given via a ruminal tube. The lambs were inspected daily and killed by being stunned by captive bolt and immediately exsanguinated on the first day that signs of photosensitization could be seen. Liver tissue was also collected from four lambs of both sexes which did not develop clinical signs of photosensitization; these lambs were killed 26 days after being dosed with 0.5 mg sporidesmin/kg body weight. Control material was collected from eight White Norwegian lambs of both sexes, 1.5-2 months old. These lambs were also stunned by captive bolt and were immediately exsanguinated. The sporidesmin-dosed lambs and controls followed their dams grazing on cultivated fields where no N. ossifragum plants grew. The lambs were killed as soon as possible after the onset of the clinical signs of photosensitization. These were increasing restlessness, head shaking, scratching of the face and ears with the hind feet, and rubbing of the irritated skin against the ground. The skin changes develop rapidly, with swelling and reddening. The eyelids, muzzle and lips become swollen and turgid. However, the most obvious signs were the thickened, oedematous, heavily drooping ears. Soon there is seepage of sticky honey-coloured serum from the thickened skin. After l-2 days this forms extensive scabs that mat the covering hair. Tissues from the gallbladder area of the liver were futed in 10% buffered formalin, embedded in paraffin wax and processed routinely for light microscopy. Sections of tissues stained with haematoxylin and eosin (HE), van Gieson and periodic acid-Schiff (PAS) stains were prepared by standard methods. Diastase tests were performed to verify that the PAS-positive material was glycogen (Moewis, 1978). The histological findings were divided into two groups, one describing the grade of portal tibroplasia and bile duct proliferation and one describing the grade of damage in hepatocytes. The findings were blindly graded on a semi-quantitative scale of increasing injury: 0, normal; 1, minor damage; 2, moderate damage; 3, severe damage. When estimating the grade of liver damage we compared our specimens with the cellular damage seen in four HE-stained sections from Panicurn-intoxicated sheep, obtained from Peter Mortimer, New Zealand. All sections were compared and the section with the most severe grade of portal tibroplasia and bile duct proliferation was
445
assessed as grade 3. The grading for periportal tibroplasia and bile duct proliferation in the other sections was decided by reference to this most severe case. The grading for the damage to the hepatocytes was done in the same way. The section with the highest number of damaged hepatocytes was assessed as grade 3 and the grading for the liver parenchymal damage in the other sections was decided by reference to that section. The PAS-sections were similarly graded on a O-3 scale of increasing glycogen content and were correlated with the enzymatically determined glycogen content (Figure 1). Small pieces of tissue were cut from the ventral edge of the liver of both the sporidesmin-intoxicated and the control lambs but not from the alveld cases. They were rinsed in ice-cold KCI, transferred to 0.05 mol/L Tris-HCl, 0.15 mol/L KCl, 1 mmol/L EDTA (pH 7.4) containing 20% v/v glycerol, frozen in liquid nitrogen, and then stored at -80°C. To determine the enzyme activities and glycogen contents, these specimens were thawed at 0°C and homogenized with a Dounce all-glass homogenizer. Homogenates were centrifuged at 900 g for 10 min and the supernatants were used for the assays. Glycogen was measured according to Keppler and Decker (1974); glucose-6-phosphatase (EC 3.1.3.9) according to Baganski et al. (1974); and glycogen phosphorylase (EC 2.4.1.1) as described by Morata et al. (1982).
RESULTS The first case of photosensitization occurred 11 days after sporidesmin dosing and the last case was detected 22 days after exposure. It is not known how many days after intoxication alveld was seen. There were no macroscopic lesions in the livers of the alveld cases. Half of the livers from the sporidesmin-intoxicated, photosensitized lambs were not macroscopically changed. The livers of the other half of the sporidesmin-intoxicated, photosensitized lambs were moderately swollen and showed yellow discoloration.
Figure 1. The association between the grades of glycogen accumulation, as determined from the PAS-sections, and the percentage hepatic glycogen content, determined enzymatically. Y = 1.78X + 0.86, R = 0.93
446
TABLE I Number of lambs in each group, parenchyma
Grade
Alveld (n =6)
0 1 2 3
0 3 0 3
classified
Sporidesmin-dosed, photosensitized (n=S)
0 1 5 2
by the grade of damage to the liver
Sporidesmin-dosed, not photosensitized (n =4)
0 3 1 0
Controls (n=S)
7 1 0 0
Figure 2. A section from the liver of a lamb affected by alveld. Areas of hepatocellular damage are indicated by arrows (grade 1). H and E, x 319
The hepatocellular damage seen in both the alveld and the sporidesminintoxicated cases was of the same type and was mostly dominated by single cell necrosis rather than by large necrotic lesions developed at the confluence of necrotic hepatocytes (Table I; Figure 2). The hepatocytes were affected centrilobularly in cases with minor or moderate grades of damage. In cases with severe damage, the hepatocytes in all regions of the lobule were affected, although the centrilobular
hepatocytes were most affected. Hydropic vacuolation was seen in the hepatocytes and in some cases lipid-like vacuoles were observed. Large quantities of bile pigment were observed in the hepatocytes and bile canaliculi of lambs with photosensitization.
TABLE II Number of lambs in each group, classified by the grade of portal libroplasia duct proliferation
Grade
Alveld (n=6)
0 1 2 3
2 4 0 0
Sporidesmin-dosed, photosensitized (II = 8)
1 5 2 0
Sporidesmin-dosed, not photosensitized (n=4)
0 1 3 0
and bile
Controls (n=8)
8 0 0 0
Figure 3. A section from the liver of a sporidesmin-dosed, photosensitized Portal libroplasia and bile duct proliferation. Grade 2. H and E, x 170
lamb.
448
Figure 4. A section from the liver of a lamb affected by alveld. Crystalloid present in the bile duct epithelium. H and E, x 320
clefts are
TABLE III Number of lambs in each group, classified by the grade of glycogena accumulation
Grade
Alveld (12=6)
0 1 2 3
1 1 3 1
Sporidesmin-dosed, photosensitized (?I = 8)
0 3 3 2
Sporidesmin-dosed, not photosensitized (n=4)
3 1 0 0
Controls (n=S)
7 1 0 0
aPAS is not a specific method for demonstrating glycogen, but by using the diastase test as well it was evident that the PAS-positive material seen in our sections was glycogen (Moewis, 1978)
449
Minor to moderate portal fibroplasia and bile duct proliferation were almost always present in the livers from both the alveld cases and the sporidesmin-intoxicated cases (Table II; Figure 3). Bile ducts obstructed by scar tissue were seen in one sporidesmin-intoxicated case. Accumulation of neutrophils appeared in both the alveld cases and the sporidesmin-intoxicated cases. Accumulation of crystalloid material in bile duct epithelium was observed in one alveld case (Figure 4).
Figure 5. A section from the liver of a sporidesmin-dosed, photosensitized PAS-positive material can be seen in the centrilobular area. PAS, x 170
lamb.
Accumulated glycogen was seen in hepatocytes in the centrilobular areas (Table III). There was a border between the hepatocytes with accumulated glycogen and those with normal glycogen deposits (Figures 5 and 6). Livers from the sporidesmin-intoxicated lambs with photosensitization contained 2.7-6.4% glycogen, except in one lamb, whose liver contained 1.1% glycogen. The hepatic glycogen content in the sporidesmin-dosed lambs that did not show photosensitization varied between 0.65% and 1.7%. In all the control lambs, except one, the glycogen content of the liver was 0.54-1.4%. In one control lamb the liver tissue contained 3.1% glycogen. Unfortunately, material for determination of enzyme activities and glycogen content was not collected from the alveld cases. In %% of the cases, the grades for parenchymal damage and glycogen accumulation were the same or one grade different (Table IV). The lamb with a difference in grading of 3 was one of the alveld cases, where the hepatocytes were affected throughout the lobules yet no glycogen accumulation was seen.
450
Figure 6. A section from the liver of a control animal. PAS-positive evident in this liver. PAS, x 170
TABLE IV The differences between the grades accumulation (from the PAS-sections)
Difference in grading
3 2 1 0
of parenchymal
material
is not
damage and of glycogen
Number of lambs
1 0 13 12
The average activity of glucose-6-phosphatase in the material from all the lambs in which this was assayed (n =20) was 55 nmol.min-‘.(mg homogenate protein)(SD=7) and that of glycogen phosphorylase (a+ b) was 81 nmol.min-‘.(mg homogenate protein)(SD = 16). The a-form was 85% + 8% of the total phosphorylase activity. Liver glycogen contents were not correlated to the activities of these enzymes and sporidesmin intoxication did not appear to affect them.
4.51
DISCUSSION It was difficult to decide when the toxic effects started in the alveld cases, but the most relevant time for histological comparison seemed to be when photosensitization occurred. Consequently, the sporidesmin-intoxicated animals were killed at different times after sporidesmin dosing. However, all the sporidesmin-intoxicated cases that showed photosensitization were killed after the ears became oedematous but before seepage of serum occurred. Because of the sheep management system in Norway, with the animals grazing large areas of uncultivated fields, regular surveillance was difficult. Four of the alveld cases were killed before seepage of serum occurred, but, in two cases, scabs could be detected on the eartips. The clinical findings in these two lambs also suggested that photosensitization had started a few days before they were killed. Kellerman and Coetzer (1984) divided the hepatogenous photosensitization diseases into two main groups, one group with damage primarily to the biliary system and the other with damage primarily to the liver parenchyma. We wished to use a similar system in classifying the pathological changes seen in our material, and graded the findings by severity. An objective scale for grade of damage was difficult to work out; however, we sought to render our results comparable by grading blind and by mutual reference between the sections. Outbreaks of photosensitization occur in sheep grazing on Panicum spp. (Kellermann and Coetzer, 1984). The pathological lesions are identical with those of geeldikkop, which also have some resemblance to the lesions of alveld (Ulvund, 1984). The lesions in Panicurn-intoxicated sheep can well be compared with the lesions seen in alveld and experimental sporidesmin intoxication. The portal fibroplasia and bile duct proliferation in Mortimer’s sections from Panicurn-intoxicated sheep were severe and assessed as grade 3. The portal fibroplasia and bile duct proliferation were not as severe in any of our alveld or sporidesmin-intoxicated lambs, the worst being assessed as grade 2. The histological changes in sporidesmin-intoxicated animals have been well studied. Our findings of portal fibroplasia and bile duct proliferation agree with previous reports (Mortimer, 1963; Kellerman and Coetzer, 1984). Mortimer suggested that the obstructive lesions in the bile ducts cause the retention of phylloerythrin in photosensitized animals. If obstruction of the bile ducts has to be present before phylloerythrin accumulates in sporidesmin-intoxicated animals, it is remarkable that photosensitization occurred in lambs with no or only minor histological changes in the bile ducts. Furthermore, if obstructive lesions of the bile ducts are the cause of phylloerythrin retention in facial eczema, we would expect that the non-photosensitized sporidesmin-intoxicated animals would be photosensitive, as these animals showed the greatest portal and biliary damage (Table II). Our results suggest that obstruction of the bile ducts may not be the only cause of retention of phylloerythrin in sporidesmin-intoxicated lambs. The relative lack of portal fibroplasia and bile duct proliferation or of pathological changes in alveld cases may also indicate that obstruction of bile ducts is not the cause of phylloerythrin retention. The occurrence of crystalloid material in bile ducts of alveld cases was first reported by Ulvund (1984). She suggested that the pathogenesis of alveld may be similar to the pathogenesis of geeldikkop (Kellerman ef af., 1980).
452
In sporidesmin-intoxicated sheep, Mortimer (1%3) reported marked vacuolation and unevenness in cytoplasmic staining, together with the loss of the cellular and sinusoidal outlines throughout the lobules. This was most marked in the centrilobular areas. Our findings both in alveld and in sporidesmin-intoxicated cases agree with these observations. The occurrence of more severe damage to the liver parenchymal cells of the photosensitized animals suggests that such damage is important for the retention of phylloerythrin. The glycogen accumulation in the hepatocytes of the photosensitized animals, and the normal glycogen deposits in non-photosensitized lambs, indicate a connection between glycogen accumulation and the occurrence of photosensitization. Mortimer (1963) found the glycogen content in parenchymal cells to be normal 10 days after sporidesmin dosing, while 14 days after dosing it varied between animals. In the most severely affected cases, little glycogen could be detected. Our findings in the sporidesmin-intoxicated animals that did not become photosensitized agree with Mortimer’s findings. The close association between parenchymal damage, glycogen accumulation and the occurrence of photosensitization supports the suggestion that there is a connection between glycogen accumulation and the occurrence of photosensitization. These findings also support the suggestion that parenchymal damage, rather than obstruction of the bile ducts, causes the retention of phylloerythrin seen in alveld and experimental sporidesmin intoxication. How the mechanism of glycogen accumulation is affected remains unidentified. However, the present results show that the hepatic glycogen phosphorylase and glucose-6-phosphatase activities were normal in all the affected animals. Further investigation of the possible connection between the accumulation of glycogen in the hepatocytes and the retention of phylloerythrin in the photosensitized lambs is desirable.
ACKNOWLEDGEMENTS We thank Ruakura Agricultural Centre, Hamilton, New Zealand for the sporidesmin, Peter Mortimer for sections from the livers of Panicurn-intoxicated animals, and The Agricultural Research Council of Norway for financial support for the project. The skilled technical assistance of Berit Christophersen and Birgit Roe is greatly appreciated.
REFERENCES Aas, 0. and Ulvund, M.J., 1989. Do microfungi help to induce the phototoxic disease alveld in Norway? Veteriirrary Record, 124,563 Abdelkader, S.V., Ceh, L., Dishington, I.W. and Hauge, J.G., 1984. Alveld-producing saponins. II. Toxicological studies. Acra Veterinaria Scandinavica, 25, 76-85 Baganski, ES., Foa, P.P. and Zak, B., 1974. In: H.U. Bergmeyer (ed.), Methodr ofEnzymatic Analysis, (Academic Press, New York), 876-880 FI&%yen, A., 1991. Alveld in lambs, (Dr. scient. thesis, Nomegian College of Veterinary Medicine, Oslo) Kellerman, T.S. and Coetzer, JAW., 1984. Heparogenous photosensitivity diseases in Soufh A@ica (Veterinary Research Institute, Onderstepoort, Technical Communication 193) Kellerman, T.S., van der Westhuizen, G.C.A., Coetzer, J.A.W., Roux, C., Marasas, W.F.O., Minne, J.A., Bath, G.F. and Basson, PA., 1980. Photosensitivity in South Africa. II. The experimental production of the ovine hepatogenous photosensitivity disease geeldikkop (Tribulosis ovis) by the simultaneous
4.53 ingestion of Tribulus terrestris plants and cultures of Pithomyces charfarum containing the mycotoxin sporidesmin. Onderstepoort Journal of Veterinary Research, 41,231-X1 Keppler, D. and Decker, K., 1974. In: H.U. Bergmeyer (ed.), Merho& of Elrzymuzic Analysis, (Academic Press, New York), 1127-1131 Laksesvela, B. and Dishington, LW., 1983. Bog asphodel (Narthecium ossifagum) as a cause of photosensitisation in Norway. Veterikary Record, 112,37537’8 Moewis, G., 1978. In: Histopatologisk Teknik, (Almqvist & Wikseh Forlag AB, Stockholm), 145-147,195 Morata, P., Faus, M.J., Perez-Palomo, M. and Sanchez-Medina, F., 1982. Effects of stress on liver and muscle glycogen phospholylase in rainbow trout (Salmo gairdneri). Comparative Biochemistiy and Physiology, 72B, 421-425 Mortimer, P.H., 1%3. The experimental intoxication of sheep with sporidesmin, a metabolic product of Pithomyces churtarum. IV. - Histological and histochemical examinations of orally-dosed sheep. Research in Veterinary Science, 4,166-l&5 Ulvund, M.J., 1984. Alveld og andre sjukdommer med spnptomer pg fotosensibilitet hos sau [A&Id and other photosensitivity diseases in sheep]. In: Gijiige Planter og Planteforgifininger Hos Dyr i Rogaland, (Rogaland veterinaerforening, Utstein Kloster), 23-29 (Accepted 31 July 1991)