Poisoning in Sheep

Veterinary Research Communications, 27 (2003) 53^62 # 2003 Kluwer Academic Publishers. Printed in the Netherlands

Experimental Tribulus terrestris Poisoning in Sheep: Clinical, Laboratory and Pathological Findings M.R. Aslani1*, A.R. Movassaghi2, M. Mohri1, M. Pedram1 and A. Abavisani2 Departments of 1Clinical Studies and 2Pathobiology, School of Veterinary Medicine, Ferdowsi University of Mashhad, PO Box 91775-1793, Mashhad, Iran *Correspondence Aslani, M.R., Movassaghi, A.R., Mohri, M., Pedram, M. and Abavisani, A., 2003. Experimental Tribulus terrestris poisoning in sheep: clinical, laboratory and pathological ¢ndings. Veterinary Research Communications, 27(1), 53^62 ABSTRACT Eleven native sheep, 1^2 years old, of both sexes were randomly divided into two groups, 6 sheep being allocated to the experimental group and 5 serving as controls. The sheep in the experimental group were fed 80% Tribulus terrestris and 20% alfalfa hay and wheat straw, while the control sheep were given a mixture of 40% alfalfa hay and 60% wheat straw. Clinical signs of hepatogenous photosensitivity were observed from day 11, including reddening and crust formation on the muzzle, nose, ears and eyelids, depression, weight loss, icterus, conjunctivitis, and yellow discoloration of the urine. Laboratory ¢ndings on weekly samples indicated signi¢cant di¡erences (p50.05) in white blood cell count, total plasma protein and ¢brinogen, total and direct bilirubin, blood urea nitrogen and creatinine concentrations, and aspartate aminotransferase and alkaline phosphatase activities. There were no signi¢cant di¡erences in the packed cell volume, in the neutrophil, lymphocyte or eosinophil counts, or in the serum calcium, phosphorus, potassium, sodium or chloride concentrations. At necropsy of the experimental animals, there were various degrees of generalized icterus and the livers were swollen and discolored by bile pigment. Histopathological examination revealed varying amounts of crystalloid material in the bile ducts and renal tubules, hepatocellular degeneration, biliary ¢brosis and proliferation, renal tubular necrosis and focal necrosis of cardiac muscle. Keywords: alkaline phosphatase, aspartate aminotransferase, bilirubin, caltrop, clinical signs, diagnosis, jaundice, photosensitivity, plasma protein, sheep Abbreviations: ALP, alkaline phosphatase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; PCV, packed cell volume; WBC, white blood cells

INTRODUCTION Tribulus terrestris (caltrop, puncture vine) is a prostrate annual herb belonging to the plant family of Zygophyllaceae and has a worldwide distribution. Under certain conditions, ingestion of this herb induces a hepatogenous photosensitization in sheep and goats, known as geeldikkop, tribulosis ovis or yellow big head (Henning, 1932; Van Tonder et al., 1972; Glastonbury et al., 1984). In Australia, a locomotor disorder of sheep has been reported to be associated with feeding of T. terrestris (Bourk, 1984; Bourk et al., 1992).

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Tribulosis ovis has great economic importance in South Africa, as more than 0.5 million animals may be a¡ected in a single season (Kellerman et al., 1996) and it has been intensively investigated in that country (Kellerman et al., 1980). It has also been reported from Australia (Bourk, 1983; Glastonbury et al., 1984; Jacob and Peet, 1987), the United States (McDonough et al., 1994), Argentina (Tapia et al., 1994) and Iran (Amjadi et al., 1977). The disease is one of the photosensitization diseases of small ruminants, characterized by the deposition of optically active, birefrigent, crystalloid material in the biliary system (Miles et al., 1994a). The occlusion of the bile ducts by crystalloid material results in failure of the liver to excrete phylloerythrin (a photodynamic porphyrin) in the bile, resulting in its accumulation in the blood (Kellerman et al., 1996). It has been suggested that the crystalloid-inducing factors in T. terrestris are the steroidal saponins diosgenin and yamogenin, which are converted to epismilagenin and episarsasapogenin following ingestion of the plant by ruminants (Miles et al., 1994a,b). It has also been shown that there is a variation in the chemotypes of saponins from T. terrestris collected from di¡erent regions (Wilkins et al., 1996). The plant also contains a mixture of the b-carboline alkaloids: harmane, norharmane, tetrahydroharmane, harmine, harmaline, harmol, harmalol, ruin and dihydroruin (Bourke et al., 1990). It has been suggested that it is these alkaloids that cause a locomotor disorder in sheep associated with prolonged ingestion of large quantities of the plant (Bourke, 1987; Bourke et al., 1992). Although T. terrestris grows in most regions of Iran, particularly in the central and north-east provinces, there is only one report of photosensitization in sheep associated with its ingestion (Amjadi et al., 1977). The plant is widely distributed in Khorasan and there have recently been a number of undocumented reports of photodermatitis thought to be due to grazing on the plant and observed by farmers and/or local veterinarians. The object of this study was to evaluate the ability of T. terrestris growing in Khorasan province to cause photosensitization in sheep and to describe the clinical, laboratory and pathological ¢ndings of the resulting disease. MATERIALS AND METHODS Animals Eleven, 1^2 year old, native and apparently healthy sheep of both sexes were dewormed 14 days before commencement of the experiment by subcutaneous injection of ivermectin (Ivomec, Razak Co., Tehran, Iran) and oral administration of rafoxanide (Ranide, Damloran Co., Tehran, Iran) at dose rates of 0.22 and 7.5 mg/kg body weight, respectively. The animals were sheared and randomly divided into two groups, 6 sheep being allocated to the experimental group and 5 sheep serving as controls. The animals were placed in two adjoining pens without protection against the prevailing climatic conditions.

55

Plant material T. terrestris, in the £owering to seeding stage, was collected in August 1998 in Sabzevar district of Khorasan province, air-dried, transported to the farm of the School of Veterinary Medicine of Mashhad and stored away from sunlight for use during the experiment. Haematology and serum biochemistry In both groups of sheep, weekly blood samples for haematological studies were taken from the jugular vein into vacutainers containing EDTA as an anticoagulant from day 0 to day 42. For the serum biochemical analyses, blood samples were collected at the same times into vacutainers and the serum was separated by centrifugation. Haematological measurements were made by standard manual methods (Dacie and Lewis, 1984) within 12 h of collection. Di¡erential leukocyte counts were performed on routinely prepared Giemsa-stained blood ¢lms using the cross-sectional technique (Jain, 1993); 100 leukocytes were identi¢ed. Serum total protein was determined by refractometry (Erma Clinical Refractometer, Erma, Tokyo, Japan). Fibrinogen concentrations were measured by a heat precipitation method (Jain, 1986). The levels of blood urea nitrogen (BUN) were measured by the diacetyl monoxime method; creatinine by the Ja¡e¨ method; total and direct bilirubin by the Jendrassik method; calcium by an o-cresolphthalein method; inorganic phosphorus by the ammonium molybdate method; aspartate aminotransferase (AST) by the Reitman and Frankel method; alkaline phosphatase (ALP) by the modi¢ed Bowers and Mecomb method; and chloride by a colorimetric (mercuric nitrate) method using a spectrophotometer (Jenway 6105, Dunmow, UK). Sodium and potassium concentrations in serum were determined by a £ame photometric method (Flame Photometer, PFP 7 Clinical, Jenway). All the enzymatic activities were measured at 378C and the results are presented in U/L. All the biochemical procedures were conducted as described by Burtis and Ashwood (1994). Feeding regimes, clinical examination and sampling The sheep in the experimental group were fed ad libitum on an 80% T. terrestris and 20% alfalfa hay mixture with chopped wheat straw (40% and 60%, respectively), while the control sheep received only the same mixture of alfalfa hay and chopped wheat straw. These feeding regimes were continued for 6 weeks. The animals were closely observed twice daily for any signs of illness or behavioural changes. They were also examined clinically at weekly intervals for their vital signs and/or abnormalities.

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Pathology The sheep in the experimental group were killed by exsanguination and necropsied on day 42. Tissue samples were collected from the liver, gallbladder, kidney, urinary bladder, heart, lung, lymph nodes, spleen, rumen, abomasum, cerebrum, cerebellum, spinal cord, skin and small and large intestines. The collected tissues were immediately ¢xed in phosphate-bu¡ered 10% formalin, embedded in para¤n wax, sectioned at 5 mm and stained with haematoxylin and eosin. Statistical analyses To evaluate the signi¢cance of the di¡erences between the mean values observed during the experiment, statistical analyses were performed using Student's t-test and analysis of variance (ANOVA) with the multiple range Duncan test; p50.05 was considered signi¢cant. RESULTS Clinical ¢ndings The sheep in the experimental group showed di¡erent degrees of clinical manifestations. The ¢rst signs were pruritis on the face, mild reddening of the eyelids and a tendency to seek shade in one sheep on day 11 and in two other animals on day 13. During the third week, three other sheep in this group gradually developed clinical signs associated with photosensitivity, including a serous ocular discharge, conjunctivitis, icterus, erythema, crust formation on the muzzle, ears and eyelids, hyperpnoea, reduced appetite, depression, weakness, discoloration of the urine and weight loss. The severity of these signs di¡ered, being most evident in three animals. No signi¢cant di¡erences were observed between the two groups of sheep in body temperature or in their heart or respiratory rates during the experiment. Laboratory ¢ndings There were no signi¢cant di¡erences in the PCV, neutrophil, lymphocyte, monocyte or eosinophil counts between the experimental and control groups. The total numbers of WBC were signi¢cantly increased (p50.05) in the experimental group in weeks 5 and 6. The total serum protein concentration increased gradually in the animals of experimental group from 64+4.6 g/L on day 0 to 78+6.4 g/L on day 42. The di¡erence was signi¢cant (p50.001) by weeks 5 and 6. The plasma ¢brinogen concentration also increased in the experimental group during the feeding of T. terrestris and there was a signi¢cant di¡erence (p50.05) between the groups in week 6.

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The serum AST and ALP activities increased in the animals in the experimental group during the experiment (p50.01). Total and direct bilirubin concentrations also increased signi¢cantly (p50.05) in the sheep in the experimental group. The serum bilirubin concentration was su¤ciently elevated in some sheep to cause yellow discoloration of the serum samples. The serum creatinine concentration in the animals in the experimental group increased gradually during the experiment and there was a signi¢cant di¡erence (p50.05) in week 6. The BUN in the serum showed a more marked and earlier elevation and di¡erences were signi¢cant (p50.05) from week 3 to week 7 (Table I). The concentrations of Ca, P, Cl, Na and K in the serum showed no signi¢cant di¡erences between the experimental and control groups. Pathological ¢ndings At necropsy, all the animals in the experimental group showed various degrees of generalized icterus. Their livers were swollen and discolored by bile pigments. The gallbladders were enlarged and ¢lled with concentrated bile. The kidneys were somewhat swollen and yellow to green in colour. Some animals showed dermatitis with facial scaling and crusts. Histological examination of the livers revealed varying amounts of crystalloid materials in the bile ducts (Figure 1) and some hepatocellular degeneration. In several of the a¡ected animals, there was also biliary ¢brosis, bile duct proliferation and in¢ltration of mononuclear in£ammatory cells. Other lesions included tubular necrosis in the kidneys, with the presence of crystalloid materials in the tubules (Figure 2), focal necrosis of cardiac muscle and epithelial necrosis of the gallbladder, accompanied by in¢ltration of mononuclear in£ammatory cells. There was pustule formation, ¢brinopurulent exudate, cell debris and in¢ltration of in£ammatory cells in the dermis of the a¡ected animals. No histological changes were found in other tissues. DISCUSSION It is clear that T. terrestris growing in Sabsevar district of Khorasan province of Iran is capable of causing hepatogenous photosensitization in sheep. Although there have been several reports of successful experimental induction of this disease by feeding T. terrestris to sheep, most experimental trials have failed to produce the disease (Henning, 1932; Van Tonder et al., 1972; Glastonbury et al., 1984; Jacob and Peet, 1987). Biliary deposition of crystalloid materials is thought to be involved in the pathogenesis of poisoning by T. terrestris and it has been suggested that the factors responsible for the formation of such occlusive materials are the steroidal saponins present in the plant (Kellerman et al., 1991). A variety of saponins have been isolated from T. terrestris, including diosgenin, yamogenin, epismilagenin, tigogenin, neotigogenin, gitogenin and neogitogenin in the ratio 10:7:1:11:7:35:25 (Miles et al., 1994a). Evidence

S

S

S

6.32+1.5

8.96+1.61

8.82+1.96

10.75+2.82* 7.28+1.04 S

11.07+3.99* 7.32+1.46 NS

Week 2 p-value

Week 3 p-value

Week 4 p-value

Week 5 p-value

Week 6 p-value

^ 99.01+19.45

^

S

NS

NS

S

84.86+14.14

80.44+14.14

94.59+17.68

72.26+19.45

110.5 +32.71 NS

NS

155.58+51.27*

115.80+60.11

114.04+21.22

97.24+8.84

101.66+18.56

84.86+12.38

^

Creatinine (mmol/L) ööööööööööö Test Control

*Signi¢cant di¡erence with ¢rst sampling time (p50.05, within groups)

NS, not signi¢cant; S, signi¢cant di¡erence (p50.05, between groups)

4.78+0.96

6.64+1.21

4.46+0.61

4.82+0.99 NS

5.32+1.61

Week 1 p-value

5.57+0.93 NS

5.07+1.54

Day 0 p-value

BUN (mmol/L) ööööööööööö Test Control

NS

S

NS

NS

NS

19.49+3.25

13.68+5.13

17.1 +5.64

7.70+7.35

9.75+5.47

32.49+5.81* 19.15+3.93 NS

25.65+12.31* 15.39+3.93 NS

23.94+3.93

21.38+3.59

19.32+4.79

8.55+4.79

7.01+2.74

Total bilirubin (mmol/L) ööööööööööö Test Control

10.43+6.67* 3.42+2.05 S

10.43+6.67* 2.74+0.90 NS

3.08+2.74 3.42+2.05 NS

3.93+1.37 4.45+3.93 NS

5.13+4.10 3.93+1.71 NS

2.91+2.91 3.93+5.30 NS

3.08+2.22 2.05+0.86 NS

Direct bilirubin (mmol/L) ööööööööööö Test Control

80.7+1.5

88.0+9.4 NS

78.6+4.1 NS

81.4+1.5 NS

80.7+1.2 NS

S

106.6+21.3* 87.4+7 NS

107.1+11.2* 90.8+7.4 S

90.0+6.5

80.0+6.5

80.1+0.9

75.7+6.8

61.5+15.4

AST (IU/L) öööööööööö Test Control

TABLE I The mean(+SD) of biochemical parameters in the serum of experimental and control groups of sheep during the study

^

28.0+7.1

20.8+1.9

22.8+13.6

21.5+3.0

^

66.1+28.9* 36.4+12.4 NS

61.8+28.5* 36.2+11.3 NS

S

S

NS

NS

48.6+15.5

40.8+5.1

33.0+7.6

24.8+8.1

^

ALP (IU/L) ööööööööö Test Control

58

59

Figure 1. Chronic cholangitis associated with peribiliary ¢brosis and plugging of the bile duct by crystalline material. H&E, 6640

has also been presented that indicates that only saponins capable of being metabolized to epismilagenin and episarsasapogenin are lithogenic, and the biliary crystals in the sheep with experimentally induced geeldikkop have been shown to be composed of a 6:1 mixture of the calcium salts of the b-d -glucuronides of epismilagenin and episarsasapogenin (Miles et al., 1994a,b). Accumulation of birefringent crystalloid material (microliths) is the most conspicuous histopathological feature of T. terrestris poisoning, and the principal occluding mechanism responsible for retention of phylloerythrin. An increase in the concentration of circulating phylloerythrin can result in photosensitization (Row, 1989). On the other hand, both lithogenic and non-lithogenic populations of T. terrestris have been identi¢ed in South Africa, because of di¡erences in the types of saponins they contain, and it is thought that this di¡erence may be determined by genetic or environmental factors (Wilkins et al., 1996). Seasonal variations in the concentration of the saponins have also been reported in the leaf and stem of Chinese T. terrestris (Wang and Lu, 1991). T. terrestris collected in August in Khorasan province of Iran could produce the disease and was lithogenic. Di¡ering results in previous experiments (Henning, 1932; Van Tonder et al., 1972; Jacob and Peet, 1987) may have been due to seasonal variations in the saponins in the plants or to di¡erent chemotypic populations of the plant.

60

Figure 2. Extensive necrosis of convuluted tubules with crystalloid materials and acicular clefts. H&E, 6640

Although the ¢ndings, which included icterus, elevation of serum AST and ALP and of total and direct bilirubin, as well as the histopathological lesions in the sheep that had received T. terrestris, indicated liver lesions and particularly a biliary disorder, the clinical manifestations of photosensitization were generally mild in comparison to other reports (Van Tonder et al., 1972; Glastonbury et al., 1984; McDonough et al., 1994; Tapia et al., 1994). This may be related to the time of the experiment, which was performed in October and November, when there are shorter days and a lower intensity of sunlight. Such conditions may not have provided enough irradiation to cause severe photodermatitis. Natural cases of tribulosis ovis have usually been reported to occur in hot and sunny seasons (Van Tonder et al., 1972; Tapia et al., 1994; Kellerman et al., 1996). Moreover, the individual susceptibility of the animals and the varying levels of circulating photodynamic agents and bile pigments may have in£uenced the occurrence and severity of the disease. Various forms of the disease, from mild to severe, have been observed in both natural and experimental cases (Van Tonder et al., 1972). The elevations in the concentrations of BUN and creatinine following feeding of T. terrestris are evidence of renal lesions, which is in agreement with previous reports (McDonough et al., 1994). From the histopathological ¢ndings, in addition to the hepatic lesions, there was renal tubular necrosis, with deposition of crystalloid

61

materials in the tubules. These ¢ndings are in agreement with those of other reports (Van Tonder et al., 1972; Amjadi et al., 1977; Bourke, 1983; Glastonbury et al., 1984). Cardiac lesions have occasionally been reported in cases of disease induced by T. terrestris (Van Tonder et al., 1972) and in diseases caused by ingestion of other plants that contain saponins. It has been suggested that the saponins are toxic to the myocardium (Dollahite et al., 1977; Bridges et al., 1987). Further studies are necessary to determine whether there are natural cases of T. terrestris poisoning in Iran and its economic importance. ACKNOWLEDGEMENTS The authors are grateful for the advice given by Dr James R. Thompson, DVM, MS in revising this manuscript. REFERENCES Amjadi, A.R., Ahourai, P. and Baharsefat, M., 1977. First report of geeldikkop in sheep in Iran. Archives of the Institute of Razi, 29, 71^78 Bourke, C.A., 1983. Hepatopathy in sheep associated with Tribulus terrestris. Australian Veterinary Journal, 60, 189 Bourke, C.A., 1984. Staggers in sheep associated with the ingestion of Tribulus terrestris. Australian Veterinary Journal, 61, 360^363 Bourke, C.A., 1987. A novel nigrostriatal dopaminergic disorder in sheep a¡ected by Tribulus terrestris staggers. Research in Veterinary Science, 43, 347^350 Bourke, C.A., Carrigan, M.J. and Dixon, R.J., 1990. Upper motor neuron e¡ects in sheep of some betacarotine alkaloids identi¢ed in zygophyllaceous plants. Australian Veterinary Journal, 67, 248^251 Bourke, C.A., Stevens, G.R. and Carrigan, M.J., 1992. Locomotor e¡ects in sheep of alkaloids identi¢ed in Australian Tribulus terrestris. Australian Veterinary Journal, 69, 163^165 Bridges, C.H., Camp, B.J., Livingston, C.W. and Bailey, E.M., 1987. Kleingrass (Panicum coloratum L.) poisoning in sheep. Veterinary Pathology, 24, 525^531 Burtis, C.A. and Ashwood, E.R., 1994. Tietz Textbook of Clinical Chemistry, 2nd edn, (W.B. Saunders, Philadelphia), 735^888 Dacie, J.V. and Lewis, S.M., 1984. Practical Haematology, (Churchill Livingstone, Edinburgh), 22^49 Dollahite, J.W., Younger, R.L. and Jones, L.P., 1977. Photosensitization in lambs grazing kleingrass. Journal of the American Veterinary Medical Association, 171, 1264^1265 Glastonbury, J.R.V., Doughty, F.R., Whitaker, S.J. and Sergeant, E., 1984. A syndrome of hepatogenous photosensitization resembling geeldikkop in sheep grazing Tribulus terrestris. Australian Veterinary Journal, 61, 314^316 Henning, M.W., 1932. Animal Diseases in South Africa, vol. II, Virus and De¢ciency Diseases, Plant Poisonings, (Gengra New Agency, South Africa), 855^867 Jacob, R.H. and Peet, R.L., 1987. Poisoning of sheep and goats by Tribulus terrestris (caltrop). Australian Veterinary Journal, 64, 288 Jain, N.C., 1986. Schalm's Veterinary Haematology, 4th edn, (Lea and Febiger, Philadelphia), 321^322 Jain, N.C., 1993. Essentials of Veterinary Haematology, (Lea and Febiger, Philadelphia), 66^67 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, P.A., 1980. Photosensitivity in South Africa. II. The experimental production of ovine hepatogenous photosensitivity disease geeldikkop (Tribulosis ovis) by the simultaneous ingestion of Tribulus terrestris plants and culture of Pytomyces chartarum containing the mycotoxin sporodesmin. Onderstepoort Journal of Veterinary Research, 47, 231^261

62 Kellerman, T.S., Erasmus, G.L., Coetzer, J.A.W., Brown, J.M.M. and Maartens, B.P., 1991. Photosensitivity in South Africa. VI. The experimental induction of geeldikkop in sheep with crude steroidal saponins from Tribulus terrestris. Onderstepoort Journal of Veterinary Research, 58, 47^53 Kellerman, T.S., Naude, T.W. and Fourie, N., 1996. The distribution, diagnoses and estimated economic impact of plant poisonings and mycotoxicoses in South Africa. Onderstepoort Journal of Veterinary Research, 63, 65^90 McDonough, S.P., Woodbury, A.H., Galey, F.D., Wilson, D.W., East, N. and Bracken, E., 1994. Hepatogenous photosensitization of sheep in California associated with ingestion of Tribulus terrestris (puncture vine). Journal of Veterinary Diagnosis and Investigation, 6, 392^395 Miles, C.O., Wilkins, A.L., Erasmus, G.L. and Kellerman, T.S., 1994a. Photosensitivity in South Africa. VIII. Ovine metabolism of Tribulus terrestris saponins during experimentally induced geeldikkop. Onderstepoort Journal of Veterinary Research, 61, 351^359 Miles, C.O., Wilkins, A.L., Erasmus, G.L., Kellerman, T.S. and Coetzer, J.A.W., 1994b. Photosensitivity in South Africa. VII. Chemical composition of biliary crystals from a sheep with experimentally induced geeldikkop. Onderstepoort Journal of Veterinary Research, 61, 215^222 Row, L.D., 1989. Photosensitization problems in livestock. Veterinary Clinics of North America: Food Animal Practice, 5, 301^323 Tapia, O.M., Giordanc, M.A. and Gueper, H.G., 1994. An outbreak of hepatogenous photosensitization in sheep grazing Tribulus terrestris in Argentina. Veterinary and Human Toxicology, 36, 311^313 Van Tonder, E.M., Basson, P.A. and Van Renburg, I.B.G., 1972. Geeldikkop: experimental induction by feeding the plant Tribulus terrestris L. (Zygophyllacae). Journal of the South African Veterinary Association, 43, 363^373 Wang, Y. and Lu, Y., 1991. Study on the quantitative analysis for the saponin in Tribulus terrestris L. Yauwu Fenzi Zazhi, 11, 70^73 Wilkins, A.L., Miles, C.O., De Kock, W.T., Erasmus, G.L., Basson, A.T. and Kellerman, T.S., 1996. Photosensitivity in South Africa. IX. Structure elucidation of a b-glucosidase-treated saponin from Tribulus terrestris, and the identi¢cation of saponin chemotypes of South African T. terrestris. Onderstepoort Journal of Veterinary Research, 63, 327^334 (Accepted: 14 June 2002)