Effects of Ipomoea carnea on the liver and on serum enzymes in young ruminants

J. COMP.PATH. 1973. VOL.~~.

EFFECTS OF AND ON SERUM

531

IPOMOEA

CARNEA

ENZYMES

IN

ON YOUNG

THE LIVER RUMINANTS

BY

S. E. I. ADAM Fact&

and G. TARTOUR

Departments of Clinical Studies and Pathology, of Veterinmy Science, University of Khartoum, Sudan and

H. M. OBEID

and 0. F. IDRIS

Sections of Pathology and Nutrition, Veterinary Research Laboratories, Ministry of Natural Resources,Sudan INTRODUCTION

The toxicity of plants to farm animals has been investigated in many countries including Great Britain, United States of America and Australia. For example, experimental feeding of certain members of the families Compositae, Leguminosae and Verbenaceae to animals has produced a wide spectrum of liver lesions and dysfunction (Thorpe and Ford, 1968; Ford, Ritchie and Thorpe, 1968 ; Gopinath and Ford, 1969; Adam, 197 1). Toxicity is ascribed to pyrrolizidine alkaloids in the families Compositae and Leguminosae (Bull, Culvenor and Dick, 1968) and to the polycyclic triterpenoid Lantadene A in the Verbenaceae (Seawright, 1963). In tropical and subtropical countries, drought and the acute shortage of grass on pastures are frequent and these conditions may force animals to consume varying quantities of poisonous plants which can cause liver damage. From time to time additional species of plants are discovered to be toxic to animals. Ipomoea carnea, a member of the family Convolvulaceae, is common throughout Central and Northern Sudan. A description of the distribution and habitat of the plant and the occurrence of toxicity in goats have been given in a preliminary report (Idris, Tartour and Adam, in press). The present work describes in detail the sequential changes in liver histology and correlates them with changes in the concentrations of enzymes in the serum of goats, sheep and calves fed with Ipomoea carnea. MATERIALS

AND

METHODS

Administration of Ipomoea carnea. Ten young Nubian goats of both sexes 4 to 30 months old, three male Desert sheep 15 to 30 months of age and two female Zebu calves 8 to 11 months old were used in the experiment. They were kept in pens at the Veterinary Research Laboratories in Khartoum and fed on lucerne and hay. The animals were each given daily 5 g. of finely divided, fresh Ipomoea leaves per kg. body weight as a suspension in water by drench. The animals were bled on a number of occasions before dosing commenced and twice weekly thereafter. Histological methods. Liver biopsies as described by Ford and Boyd (1962) were obtained before the first dose of the plant and then at weekly intervals throughout the experiment. The core of liver was drained on filter paper and immediately fixed

532

s. E. ADAM

et al.

in 10 per cent. formol-saline. Paraffin sections were stained with haematoxylin and eosin (H. & E.), Masson’s trichrome, Gordon and Sweet’s method and the periodic acid-schiff (PAS) method with and without incubation with diastase. Chemical methods. Blood samples were allowed to clot and the separated sera were stored at -20 “C. until analysed. Activities of aspartate aminotransferase (E.G. 2.6.1.1; G.O.T.) and alanine aminotransferase (E.C. 2.6.1.2 G.P.T.) were measured by the method of Reitman and Frankel (1957). Arginase (E.C. 3.5.3.1.) was measured by the method of Cornelius and Freedland (1962). RESULTS

Clinical

Response to Ipomoea Administration

Goats. Goats 1, 2 and 3 showed anorexia, dullness and depression on day 13. Goat 1 died 2 days later. Goat 2 became completely inappetent, reluctant to move and dyspnoeic, 22 days after the commencement of dosing. It died on day 25. Goat 3 regained appetite for lucerne and hay by day 16, but showed dullness and partial paresis of the hind limbs 10 days later. It died on day 33. By 36 days, goats 4 and 5 became anorexic and dull, and developed abduction of the hind limbs and staggering. They died on days 38 and 39 respectively. Goat 6, partially lost appetite for lucerne and hay on day 40 and died by day 49. Goat 7, developed respiratory distress, pallor of the visible mucous membranes and paresis of the hind limbs on day 100, and died on day 107. Goats 8, 9 and 10, gradually became anorexic from day 90 to day 12 1. They showed pallor of the mucous membranes, abduction and paresis of the hind limbs. They were killed on day 12 1. Sheep. The symptoms produced by Ipomoea carnea in sheep differed only in degree. Sheep 1 showed profound depression, anorexia, arching of the back and weakness of the hind limbs accompanied by staggering on day 3. Dyspnoea and recumbency followed on day 4 and death occurred on day 5. In sheep 2 and 3 the principal symptoms which appeared within 40 to 50 days of ingestion of the first dose of the plant were depression, dullness and inappetence. Thirty days later, both animals showed pallor of the visible mucous membranes and wasting. Sheep 2 died without any further associated signs on day 102 and sheep 3 was killed on day 12 1. Calves. The clinical response to Ipomoea dosing differed in the 2 calves. In calf 1, 76 days after administration commenced, the animal appeared dull, and appetite for lucerne and hay was reduced. Forty five days later i.e. 121 days after dosing commenced, the calf was killed in a weak condition. In calf 2, no obvious abnormalities were observed during the course of the experiment, apart from partial refusal of lucerne and hay during the 30 to 40 days period of the experiment. It was killed and examined post-mortem on day 12 1. The weights of the goats, sheep and calves, the daily dose and the total amount of Ipomoea they received and the time of death are given in Table 1. Post-mortem Findings Goats. In goat 1, small amounts of blood were found in the peritoneal cavity and in the mesentery. The spleen was soft and both kidneys were congested. and the abomasal mucosa and the The enlarged liver was haemorrhagic,

F F F M F

F ii k M M F F

1 2 3 4 i

7 : .10 Sheep 2 3 Calf 1 2

Goat

4

18 12 15 .~ 15 24 30 1:

3: 12

::

; 5

5 5

i:5

5 5 5 5 z

9.49 12.55 16.03 27.68 45.29

IO.39 IO.18 9.76

0.79 2.32 3.23 2.43 4.3 3.541

Total Daily amount amount of of Ipomoea Ipomoea Age Species kb. Sex months given (g./kg.) given (kg.)

SURVIVAL

AND

BODY

23.00 13.00 25.50 15.50 22.50 16.50 17.50 17.50 28.00 31.00 49.00 77.50

22.00 12.50 27.50 15.00 26.50 13.50 17.50 17.50 29.50 27.00 44.50 71.00

Before Ipomoea --__~~~~ given 8

TIMES

17.00 28.00 32.00 48.50 77.00

20.00 17.00 17.50

20.00 13.00 24.50 15.50

15

WEIGHTS

kg.)

16.00 25.00 27.00 48.00 76.00

2 1.00 12.50 20.00 14.00 18.00 15.50 17.00

22

(in

TABLE

1 ANIMALS

DOSED

WITH

i)~omoea

&mm

13.00 22.00 14.00 17.50 17.00 15.50 15.00 24.00 28.00 47.50 75.00

30

14;o 18.00 17.00 16.00 16.50 25.50 29.00 47.00 73.00

13<0 18.00 16.00 15.00 16.00 24.00 29.00 44.00 73.00

47

-

40

7.00 5.50 15.00 i,4.50 2 5.00 2 6.50 4 6.50 76.00

-

-

55

17.50 15.00 14.00 14.50 22.00 27.00 46.00 73.00

63

IS-00 16.00 14.50 14.00 21.00 28.00 45.00 74.00

-

71

Weights of animals (in kg.) during the experiment at days after Ipomoea given

OF

16.00 14.50 15.00 13.00 22.00 23.00 44.50 76.50

-

79

At death

14.70 13.65 13.65 23.10 42.20 73.40

1:; 121 121 121 102 121 (killed) 121 (killed) 121 (killed)

-- ii: -- E - 39 -

__-______ I13

534

S. E. ADAM et al.

subendocardium revealed ecchymoses and/or petechiae. The lungs were focally oedematous and the brain was congested. In goats 2 and 3, some similar changes were seen as in goat 1. Additional findings were hydrothorax and hydropericardium. In goats 4, 5 and 6, the macroscopic lesions were similar to those found in the previous goats, but hydrothorax, congestion of the kidneys and petechiae in the subendocardium were less marked. The liver was pale and showed small necrotic foci. In goat 7, the main gross lesions were focal hepatic necrosis, hydroperitoneum, ecchymotic haemorrhages at the corticomedullary junction of both kidneys, flabbiness of the heart and oedema of the lung. In goats 8, 9 and 10, the pale liver had a slightly irregular surface and was focally fibrotic. Ascites, hydropericardium and hydrothorax were less marked than those seen in the previous goats. Sheep.In sheep 1, small quantities of blood were found in the peritoneal and thoracic cavities. The lungs, brain and kidneys were slightly congested. The heart was flabby and the liver was pale and soft in consistency. In sheep 2 and 3, the gross lesions were more or less similar. Small quantities of strawcoloured fluid were found in the peritoneal, pericardial and thoracic cavities. Pulmonary oedema and congestion of the kidneys particularly at the corticomedullary junction were seen. The liver was firm and showed scattered pale spots on the surface. Calves. In calf 1, the macroscopic lesions were similar to those found in sheep 2 and 3. Hydroperitoneum, hydropericardium and hydrothorax were more marked, but little congestion was seen in the lungs and kidneys. The liver was paler and had a soft consistency. Calf 2 showed no significant gross lesions in any organ or tissue. Histological Changes Goats. In goats 1, 2 and 3, there was an overall reduction in cytoplasmic

basophilia of the hepatocytes on day 7. Congestion of the sinusoids with focal haemorrhages in the parenchyma, varying degrees of cytoplasmic fatty vacuolation and depletion of glycogen were seen on day 15. The fatty change in the inner parts of the lobules persisted, and there was infiltration with mononuclear cells and a few polymorphonuclears in the portal areas of goats 2 and 3 on day 22. Fatty change, focal hepatocellular necrosis and marked mononuclearcell infiltrations in the portal areas of goat 3 were seen on day 29. In goats 4, 5 and 6, there was loss of glycogen from the vacuolated cells in the liver biopsy samples removed on days 15 and 22. The portal tracts were enlarged and contained increased numbers of mononuclear cells, new bile ductule formation and periductal accumulation of fibroblasts on day 29. There was focal parenchyma1 cell necrosis and an accumulation of mononuclear cells and fibroblasts in goat 6 by day 44. In goats 7, 8, 9 and 10, fatty cytoplasmic vacuolation and depletion of glycogen from the hepatocytes were slight in the biopsy samples obtained on days 15 and 22, but isolated necrotic cells and infiltration with mononuclear cells and polymorphonuclears in the portal tracts were seen. Congestion of the sinusoids and focal haemorrhages in the parenchyma were observed on day 29. There were focal increases of bile ductules, and fibroblasts were seen on days 44 and 51. The biopsy specimens obtained between

EFFECTSOF Ipomoea carnea 0~ RUMINANTS

535

days 58 and 107, showed focal hepato-cellular necrosis and portal fibroplasia. The portal fibrosis was due to the deposition of mainly collagenous fibres. Goat 7 died on day 107. Sheep. Sheep 1 died on the 5th day of the experiment. When examined post-mortem the liver showed widespread cytoplasmic fatty vacuolation of the hepatocytes, necrosis of isolated cells and a reduction in the glycogen content of the affected cells. In sheep 2 and 3, liver biopsies taken from both sheep at 7 and 14 days, showed a slight overall reduction of cytoplasmic basophilia of the parenchymal cells. On the 21st day, the centrilobular and the midlobular parenchymal cells of the liver showed fatty changes. There was loss of glycogen from affected cells. There were isolated necrotic cells in the liver of sheep 2. The biopsies obtained during the period 28 to 45 days revealed an accumulation of mononuclear cells and a few polymorphonuclears in the portal tracts. Scattered vacuolated cells in the inner parts of the lobule and loss of glycogen were still seen. Slight fatty changes in the parenchyma and fibroblasts in the portal tracts were seen in the liver biopsies obtained during the period from 52 to 76 days. Focal portal fibrosis was seen on the 90th day and in the liver of sheep 2 examined post-mortem on day 102. Sheep 3 was killed 121 days after the commencement of dosing and the changes in the liver during the period horn 90 to 121 days were similar to those seen in the liver of sheep 2 examined post-mortem. Chives. On the Zlst day after dosing commenced, the liver histology in calf 1 was similar to that found in sheep 2 and 3, but a smaller number of liver cells were affected by cytoplasmic fatty vacuolation and loss of glycogen. Necrosis of isolated cells, slight fatty changes and an accumulation of mononuclear cells and polymorphonuclears in portal tracts were seen in the biopsy specimens obtained between the 28th and 60th days. Increased numbers of fibroblasts and a slight increase in portal connective tissue were seen in the liver biopsy specimens examined during the period from 76 to 99 days and at post-mortem on day 12 1. In calf 2, the lesions were less severe than those seen in the liver of calf 1 during the period from 21 to 76 days. There were fatty changes, loss of glycogen from affected cells and an accumulation of mononuclear cells, polymorphonuclears and fibroblasts in the portal tracts. Subsequent biopsies did not show progressive changes and at post-mortem on day 121 the liver was normal. Neither the brain nor the spinal cord showed any significant. histopathological changes.
536

s. E. AD.4Met al.

day 22 in the case of GOT and day 27 in the case of arginase. The concentrations of both enzymes in the serum were elevated at the time of death (Fig. 1). In goats 4, 5, 6 and 7 which lived for 38, 39, 49 and 107 days respectively after the commencement of feeding, the serum enzyme changes seen in goat 7 are typical of the group. Enzyme concentrations commenced to rise about the 10th day, reached peaks about day 32 in the case of GOT and about day 20 in the case of arginase. GOT fell to slightly above the normal level on day 40, rose to a high but fluctuating level in the 50 to 100 day period and then returned to normal at the time of death. Arginase fell gradually to normal by day 75 and remained normal until death (Fig. 2). In goats 8, 9 and 10 enzyme concentrations commenced to rise about day 15 and reached peaks about day 18 in the case of GOT and about day 20 in the case of arginase. A second rise followed in the case of GOT to a peak about day 55 and a return to normal about day 105. A third rise followed on day 110, but there was a return to normal by the 118th day. Arginase fell in a fluctuating manner to normal levels about 16 days before death (Fig. 3). Sheep.Sheep 1, died 5 days after the start of dosing, and arginase and GOT activity in the serum was high at that time. A marked increase in the activity of GOT and arginase occurred in the sera of sheep 2 and 3 (Figs. 4 and 5).

3

GPT

50 t

-30

0 t

12

24

36

48

Time (days)

Fig. 1. Serum enzyme changes in a goat dosed orally with 5 g./kg./d. of Ipomoea camea for 33 d. The arrow on the time axis indicates the commencement of dosing (as in Figs. 2 to 7).

EFFECTS

OF

i--OY?lOt?a

CaY?l.H

ON

RUMINANTS

90

, 120

537

3 -

50 3

t

-

0’.-

GPT ‘) -;tE&p~,~c.?..~~*... -30 0 30

60 Time (days)

t

Fig. 2. Serum enzyme changes in a goat dosed orally with 5 g./kg./d. of Zpomoeacamea 150

,

,

I

30

t

I

I

60

90

for 107 d.

I

120

150

Time (days)

Fig. 3. Serum enzyme changes in a goat dosed orally with 5 g./kg./d.

of Zponwea carma for 121 d.

538

s. E. ADAM

et al.

0

50 5 2

GPT t

:-~.~~5.+A..‘*~~-.w

0’

-30

0

t

30

60

90

Time (days)

Fig. 4. Serum enzyme changes in a sheep dosed orally with 5 g./kg./d. of Ipomoea carnea for 102 d.

3 L

50 F GPT

.-IIT -. r-t .- 3dL,aA+,.*.&~ ’ -30 0 30 60 90 I20 t

150

Time (days)

Fig. 5. Serum enzyme changes in a sheep dosed orally with 5 g./kg./d. of Z~omoeacarnca for 121 d.

EFFECTS

OF f,OmOea

CUT?.WaON RUMINANTS

539

3- =o GPT l 0’

-,-*w~b.4p. T

-30

0

e.di..~.q*

30

90

changes in a calf dosed orally with 5 g./kg./d. 150,

,

,

120

Time (days)

t

Fig. 6. SerumIenzyme

60

I

I

I

of Zfiamoeucarnea for 12 1 d. I

1

5

-

t

Time (days)

Fig. 7. Serum enzyme changes in a calf dosed orally with S,/kg./d. of Zpomoeacarneu for 12 1 d.

540

s. E. ADAM

et al.

The peak of activity was reached on the 22nd or the 32nd day after dosing and levels had returned to normal by the 107th day in sheep 3. In sheep 2, a normal level of arginase was reached by the 77th day whilst GOT concentrations, although fluctuating, were slightly high at the time of death. Calves. In the calves, there were rises in arginase and GOT activity within 33 or 45 days of the first dose (Figs. 6 and 7). Enzyme concentrations fell and were at pre-dosing values by the 105th day. DISCUSSION

The normality of the structure of the livers of the experimental animals was established by the examination of samples removed by needle biopsy, and the activity of enzymes was established in serial serum samples collected prior to oral dosing with fresh green leaves of Ipomoea carnea. Our results provide evidence of changes in the liver cells as well as in the portal tracts. The response of the goat’s liver to oral dosing with Ipomoea carnea commenced with a decrease in cytoplasmic basophilia in the hepatocytes, fatty change and reduced intensity of glycogen staining of the liver cells in the 7 to 15 day period. At the time when hepato-cellular necrosis developed, there were varying degrees of fatty change and depletion of glycogen from liver cells. Cell necrosis was a terminal effect of Ipomoea carnea in nearly all of the goats which lived for more than 22 days. These liver-cell changes were less severe in the calves than in goats or sheep. The simultaneous release of the intra-cytoplasmic enzymes arginase and GOT into the serum at the time when the morphological changes were apparent is further evidence of liver-cell damage. Elevated serum enzyme activity is commonly attributed to cell necrosis (Boyd, 1962 ; Ford and Lawrence, 1965 ; Thorpe, Gopinath, Jones and Ford, 1969; Ford, Adam and Gopinath, 1972) or to physical disruption of the cell wall (Cornelius and Kaneko, 1963). Ford and Lawrence (1965) and Ford (1965) correlated enzyme release with structural changes in the liver of calves and sheep which had been given either carbon tetrachloride or sporidesmin. We now confirm this finding in the goat, another ruminant, and in sheep and calves after dosing with fresh green leaves of Ipomoea carnea. Ford et al. (1968) suggested that the differing response pattern of the serum enzymes in calves fed with different levels of ragwort might lie in their localization within the cells. The failure of GPT to rise in the serum of Ipomoea-dosed animals may be attributed to a low level of this enzyme in their livers. The lack of release of this enzyme into the serum of ruminants has been previously observed, for example, in bovine acetonaemia (Ford and Boyd, 1960), seneciosis (Ford et al., 1968; Adam, 197 l), dimidium poisoning in cattle (Ford and Boyd, 1962) and in sporidesmin and lantana poisoning in sheep (Ford, 1967; Gopinath and Ford, 1969). Portal changes consisting of accumulation of mononuclear cells and polymorphonuclears, new bile duct formation and portal fibrosis were features of the response of the caprine and ovine livers to Ipomoea intoxication. In the calves, particularly in calf 2, there was regression of the hepatocellular change

EFFECTS

OF

i-pOTtlOt?a

CcWlt?a

ON

RUMINANTS

541

and the uninterrupted oral administration of Ipomoea leaves at a dose rate of I not produce portal fibrosis. One possible explanation for the 5 g./kg./day d’d difference in susceptibility of the 2 calves to Ipomoea poisoning may be the greater age of calf 2 because other factors such as breed, sex, feed, dose and route of administration were not different. The active principles contained in Ipomoea carnea are not known, but it seems likely that the toxic principles have a stimulatory effect on fibroblasts with the consequent development of portal fibroplasia. Some extrahepatic lesions in the form of haemorrhage and/or oedema were seen in the lungs, heart and kidneys at death. It was found that the continued oral administration of fresh leaves of Ipomoea carnea to goats is associated with the development of anaemia (Tartour et al., in preparation). This experiment showed that the continued administration of 5 g./kg. of fresh leaves of Ipomoea carnea is fatal in both goats and sheep, but a similar treatment may not be fatal in calves. It seems likely that the dyspnoea seen in goats resulted from pulmonary oedema. In our opinion, the nervous signs in the goats and sheep may be a consequence of hepatic damage rather than primary involvement of the central nervous system because of the absence of significant histopathological changes in the brain and the spinal cord, SUMMARY

The sequential changes in the livers of 10 goats, 3 sheep and 2 calves given daily oral doses of 5 g./kg. body weight of fresh leaves of Ipomoea carnea were studied by serial biopsy. Seven goats and 2 sheep died during the 5 to 107 day period and 3 other goats and 1 sheep were killed in a moribund state, 121 days after the commencement of dosing. Neither of the 2 calves died during the experiment. Inappetence, dullness, dyspnoea and paresis of the hind limbs were prominent clinical signs of Ipomoea poisoning in both goats and sheep. Gradual deterioration of the general condition and debility were seen in one of the calves. Both calves were killed on the 121st day. Reduction in cytoplasmic basophilia of the liver cells, fatty change, loss of glycogen, focal hepatocellular necrosis, accumulation of mononuclear cells and polymorphs, new bile ductule formation and portal fibrosis developed between the 21 and 121 days in goats and sheep. None of the calves showed hepatic portal fibroplasia. Regression of the parenchymal-cell changes was seen in one of the calves. All animals showed an increase in the concentrations of glutamate-oxaloacetate transaminase and arginase in the serum. Glutamate pyruvate transaminase activity did not change. Post-mortem examination of goats, sheep and one of the calves revealed excess fluid in the serous cavities, oedema of the lungs, congestion of the kidneys, subendocardial haemorrhage and necrosis of the liver and hepatic fibrosis. ACKNOWLEDGMENTS

We wish to thank Dr J. McC. Howell of the Faculty of Veterinary Science, University of Liverpool, for the histopathological examination of the central nervous system. Our thanks are due to Professor E. A. Karib, Dean of the Veterinary Faculty,

542

S. E. ADAM et d.

University of Khartoum and to Dr M. El Tahir, laboratories.

for his advice and encouragement Chief Veterinary Research Officer

throughout the work for the facilities of his

REFERENCES

Adam, S. E. I. (1971). Ph.D. thesis; University of Liverpool. Boyd, J. W. (1962). Res. vet. Sci., 3, 256. Bull, L. B., Culvenor, C. C. J., and Dick, A. T. (1968). The Pyrrolizidine AlkaloidsTheir Chemistry, Pathogenic@ and Other Biological Prokerties, North Holland Publishing Company; Amsterdam. Cornelius, C. E., and Freedland, R. A. (1962). Cornell Vet., 52, 344. Cornelius, C. E., and Kaneko, J. J. (1963). Cl’mica1 Biochemistry of Domestic Animals, Academic Press; New York and London. Ford, E. J. H., and Boyd, J. W. (1960). R es. vet. Sci., 1, 232, (1962). 3. Path. Bact., 83, 39. Ford, E. J. H., and Lawrence, J. A. (1965.) 3. camp. Path., 75, 185. Ford, E. J. H. (1965). Ibid., 300; (1967). Ibid., 77, 405. Ford, E. J. H., Ritchie, H. E., and Thorpe, E. (1968). Ibid., 78, 207. Ford, E. J. H., Adam, S. E. I., and Gopinath, C. (1972). Ibid., 82, 355. Gopinath, C., and Ford, E. J. H. (1969). 3. Path., 99, 75. Idris, 0. F., Tartour, G., Adam, S. E. I., and Obeid, H. M. (1973). Trap. Anim. Hlth. Prod. (in press). Reitman, S., and Frankel, S. (1957). Amer. 3. clin. Path., 28, 56. Seawright, A. A. ( 1963). Aust. vet. J., 39, 340. Thorpe, E., and Ford, E. J. H. (1968). 3. camp. Path., 78, 195. Thorpe, E., Gopinath, C., Jones, R. S., and Ford, E. J. H. (1969). 3. Path., 97, 241. [Received for @&cation,

January 9th., 19731