Pathophysiological studies on Dicrocoelium dendriticum infection in sheep

Veterinao, Parasitology, 39 (1991) 61-66 Elsevier Science Publishers B.V., A m s t e r d a m

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Pathophysiological studies on Dicrocoelium dendriticum infection in sheep Y. Theodoridis a, J.L. Duncan b'l, J.M.

M a c L e a n b a n d C.A. H i m o n a s a

aFaculty of Veterinary Medicine, Aristotelian University, Thessaloniki, 54006 Greece bFaculty of Veterinary Medicine, University of Glasgow, Glasgow, G61 1QH, UK (Accepted 8 January 1991 )

ABSTRACT Theodoridis, Y., Duncan, J.L., MacLean, J.M. and Himonas, C.A., 1991. Pathophysiological studies on Dicrocoelium dendriticum infection in sheep. Vet. Parasitol., 39:61-66. Red cell kinetics and plasma protein metabolism were investigated in two experiments using 20 adult sheep naturally infected with Dicrocoelium dendriticum, but free of other liver and gastrointestinal helminths. In the first experiment, where groups of animals with low to high Dicrocoelium burdens were injected with 5~Cr-labelled red cells and ~zSI-labelled albumin, the results indicated that there were no significant differences in the turnover rate of labelled red cells or albumin between any of the groups. In the second experiment, two groups of sheep with low and high worm burdens were studied using the same radioisotope tracers; in addition, S9Fe-citrate was used to assess red cell iron incorporation rates in the two groups. Although the red cell half-lives of the infected sheep were just significantly longer, both were within normal limits and the difference was attributed to random variation within the two small groups of sheep. No significant differences were found in the other parameters. It was concluded that burdens of up to 4000 D. dendriticum do not cause significant blood or plasma protein loss in sheep.

INTRODUCTION

Dicrocoelium dendriticum, the lancet fluke, has a wide host range and is considered to be an important parasite in sheep, goats and cattle in many countries of Europe and Asia. It occurs in the bile ducts and heavy long-standing infections cause gross induration and scarring of the liver with severe cholangitis and widespread biliary fibrosis (Mapes, 1951; Massoud, 1981; Jubb et al., 1985 ) which result in serious economic loss due to liver condemnation. Apart from this aspect of infection, little is known of its pathogenic effects. This is partly because of the complex life cycle which requires land snails and ants as intermediate hosts (Krull and Mapes, 1952, 1953), making experiAuthor to w h o m c o r r e s p o n d e n c e should be addressed.

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Y. THEODORIDIS ET 4L.

mental studies difficult. In addition, sheep in areas where infection with D. dendriticum is prevalent may also suffer from infection with more pathogenic liver flukes such as Fasciola hepatica and Fasciola gigantica (Toparlak and Gul, 1988), as well as gastrointestinal and lung nematodes; this complicates the assessment of any pathogenic effects attributable to Dicrocoelium alone. Radioisotope techniques have proved valuable in clarifying the pathogenic effects of a number of gastrointestinal and liver parasites of domestic livestock (Dargie, 1975 ). In many cases, there is an increased loss or breakdown of important body constituents such as red blood cells and plasma proteins, but these may go undetected because of compensatory mechanisms in the host. The use of 125I-labelled plasma proteins, and 51Cr- and 59Fe-labelled red cells, can reveal and quantify insidious effects of parasites. This paper describes the results of initial experiments using these techniques to study the pathogenic effects of D. dendriticum in field cases of infection. M A T E R I A L S ,AND M E T H O D S

Experimental animals" and design Twenty cross-bred adult female sheep, free from Fasciola infection but naturally infected with D. dendriticum, were used in two experiments (Table 1 ). All animals were removed from pasture, treated with invermectin or benzimidazotes to remove gastrointestinal and lung nematodes, and housed indoors for 20 days prior to the c o m m e n c e m e n t of each study. They were then placed in individual metabolic stalls and fed twice daily with a pelleted diet. Water was available ad libitum. At regular intervals throughout the experiments, TABLE 1 Experimental design Animals (naturally infected with Dicrocoelium ) Experiment 1 12 sheep previously treated with BZ

Radioisotopes Used

5~Cr-- red cells ~251 - - albumin

Experiment 2 8 sheep 5 ~ C r - red cells ~251 - - albumin G r o u p X - - 4 animals previously treated with BZ 59Fe - - transferrin and ivermectin G r o u p Y - - 4 animals previously treated with BZ, ivermectin and netobimin ( active against Dicrocoelium ) BZ = benzimidazoles.

D I C R O C O E L I U M D E N D R I T I C U M I N F E C T I O N IN SHEEP

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body weights and packed red cell volumes were monitored, and samples were obtained for serum albumin (Rodkey, 1965) and serum protein (Weichselbaum, 1940) estimations.

Worm recoveries Flukes were recovered at necropsy using the perfusion technique of Wolff etal. (1969).

Radioisotopic methods Erythrocytes were labelled with 51Cr and the circulating red cell volume and half-life (tl/2) were measured as described by Holmes et al. ( 1968 ). Ovine albumin was labelled with 125I by the method of McFarlane (1958), and plasma volume and circulating plasma albumin t l/2 were measured (Holmes and MacLean, 1971 ). Transferrin was radiolabelled with 59Fe by adding 24 Mbq 59Fe as ferric citrate to heterologous ovine plasma 60 min prior to injection (Experiment 2 only). The sheep were injected intrajugularly with 7.5 Mbq 5iCr_red cells, 3 Mbq 59Fe-transferrin and 7.5 Mbq 125I-albumin using a three-way tap assembly. Blood samples were taken at 15, 30, 60 and 240 min post-injection, and at twice daily or daily intervals for 14 days. Blood and plasma samples were assayed for 51Cr, 125I and 59Fe in a multichannel gamma spectrometer. RESULTS

The 12 sheep in Experiment 1 were divided into four groups retrospectively in relation to their D. dendriticum burdens at necropsy, and the results are presented in Table 2. Groups A and B had low burdens of < 100 and < 1000 flukes, and those of Groups C and D had 1000-2000 and > 2000 flukes, respectively. There were no significant differences in the red cell parameters of all four groups, although the mean values of the half-lives of 51Cr-labelled red cells were longest in the animals in Group A which had the lightest infections compared with those of the three other groups. The total protein and serum albumin values of all four groups were within the normal range, although the mean 125I tl/2 value was lowest in the animals with the lightest Dicrocoelium burdens. In Experiment 2, eight sheep were divided into two groups. One (Group X) had naturally acquired worm burdens of approximately 2500-4500 adult D. dendriticum, while the second (Group Y) had received, in the 3 weeks prior to the start of the radioisotope study, several treatments with netobimin (Hapadex, Schering-Plough) which resulted in negative or extremely low burdens of < 10 D. dendriticum (Table 3). In this experiment, there was a

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TABLE 2 Red cell and plasma protein indices in sheep infected with D. dendriticum (Experiment 1 ) Group

Serum albumin (gl i)

1251 tl.

74.1 80.0 84.3

34.8 32.9 31.8

328 260 288

199

79.5

33.2

292

19.60 19.70 30.80

149 160 134

70.4 94.1 85.5

41.0 34.9 34.7

314 422 405

31.3

23.37

148

83.3

36.9

380

1050 1670 1270

34.0 23.5 31.0

19.30 12.40 22.70

145 139 203

101.9 91.5 86.0

41.0 20.4 38.4

389 296 301

Mean

1330

29.5

18.13

162

93.1

33.3

329

7 10 11

3600 2040 4630

27.0 27.5 31.5

15.90 17.50 23.20

150 163 143

76.1 71.6 94.9

34.1 38.7 31.1

331 335 552

Mean

3423

28.6

18.87

152

80.9

34.6

406

Sheep No.

A

Red cell volume (mlkg t)

5~Cr t~/2 (h)

29.5 31.0 24.5

12.00 11.00 19.00

248 174 174

57

28.3

14.00

1 5 12

840 680 300

35.0 25.5 33.5

Mean

607

2 4 6 Mean

B

C

3 8 9

D

Total fluke burden

PCV

0 100 60

Total protein (gl - I )

2

(h)

PCV = packed cell volume.

TABLE 3 Red cell and plasma protein indices in sheep infected with D. dendriticum (Experiment 2) Group Sheep Total PCV No. fluke burden

Red S~Cr t]/2 cell (h) volume (mlkg L)

Total Serum Plasma ~2Sl tE/2 protein albumin volume (h) (g1-1) (gl - I ) (mlkg 1)

SgFe l~/2 (rain)

X

Y

21 22 24 25

2690 3240 4310 2430

31.0 31.0 34.0 25.5

17.63 17.32 14.01 t2.25

290 339 384 339

100.0 86.0 74.0 72.0

26.0 31.0 32.0 31.0

58.10 50.70 37.50 42.10

360 381 296 265

67.90 105.00 130.30 101.50

Mean 3163

30.4

15.30

338

83.0

30.0

47.10

326

101.18

27 28 29 32

9 1 10 0

29.5 35.5 30.0 29.5

18.25 22.41 22.26 15.91

284 223 266 281

75.0 67.0 69.0 75.0

34.0 33.0 30.0 38.0

47.00 43.30 47.60 38.40

337 292 309 279

51.80 164.90 150.40 83.00

5

31.1

19.71

264

71.5

33.8

44.08

304

112.53

Mean

PCV = packed cell volume.

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significant difference ( P < 0.05 ) in the mean 5i Cr tl/2 of the heavily infected sheep (Group X) which was 338 h compared with 264 h in the animals carrying negligible fluke burdens (Group Y). There were no significant differences between the groups in any other measurement. DISCUSSION

From the first experiment, it appeared that the animals with the heavier Dicrocoelium burdens had slightly reduced, although not statistically significant, red cell survival times, suggesting that there might be a loss of red blood cells associated with infection. This was perhaps not unexpected since this is a well-recognised feature of infection with more pathogenic blood-sucking helminths such as F. hepatica (Holmes et al., 1968 ) and Haemonchus contortus (Dargie and Allonby, 1975). The results of Experiment 1 prompted the second experiment which was designed to further investigate red cell kinetics and plasma protein metabolism in more defined groups of animals, i.e. those with negligible and moderately heavy Dicrocoelium worm burdens. There were significant differences in worm burdens in Experiment 2, i.e. a mean of more than 3000 flukes in the infected group (Group X) compared with five flukes in the netobimin-treated group (Group Y). However, with one exception, none of the parameters measured showed any differences between the two groups. The exception was the finding that the mean 51Cr-red cell half-life of the more heavily infected group was just significantly ( P < 0.05 ) longer than that of the control group. This result is difficult to explain and is probably attributable to individual variation in the two small groups of sheep studied. Such variations have previously been reported by Holmes et al. (1968 ) and may be due to sequestration of labelled cells in the spleen and their subsequent release; certainly, the 59Fe tl/2 results showed no evidence of any altered uptake of 59Fe which might have indicated changes in red cell production in the heavily infected sheep. If, however, the increase in 51Cr-red cell half-life in the infected group is genuine, it might be related to reticuloendothelial blockade, a consequence of the Dicrocoelium infection. The present conclusion from both of these experiments is that, unlike F. hepatica infection, there is no increased loss of red cells or plasma albumin due to the activities of Dicrocoelium at the various levels of infection found at necropsy, i.e.up to 4000 flukes. ACKNOWLEDGEMENTS

This work was supported by the International Atomic Energy Agency, Vienna, and the Ministry of Research and Technology, Greece.

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Dargie, J.D. and Allonby, E.W., 1975. Pathophysiology of single and challenge infections of Haemonchus contortus in Merino sheep: studies on red cell kinetics and the ~self-cure' phenomenon. Int. J. Parasitol., 5: 147-157. Holmes, P.H. and MacLean, J.M., 1971. The pathophysiology of ovine ostertagiasis: a study of the changes in plasma protein metabolism following single infections. Res. Vet. Sci., 12: 265271. Holmes, P.H., Dargie, J.D., MacLean, J.M. and Mulligan, W., 1968. The anaemia in fascioliasis. Studies with 5~Cr-labelled red cells. J. Comp. Pathol., 78: 415-420. Jubb, K.V.F., Kennedy, P.C. and Palmer, N., 1985. Pathology of Domestic Animals. Academic Press, London, 286 pp. Krull, W.H. and Mapes, C.R., 1952. Studies on the biology of Dicrocoelium dendrilicum (Rudolphi, 1819; Looss, 1899; Trematoda:Dicrocoeliidae), including its relation to the intermediate host. Cionella lubrica (Muller). VII. The second intermediate host of Dicrocoelium dendriticum. Cornell Vet., 42: 603-604. Krull, W.H. and Mapes, C.R., 1953. Studies on the biology of Dicrocoelium dendrilicum (Rudolphi, 1819; Looss, 1899; Trematoda:Dicrocoeliidae), including its relation to the intermediate host, Cionella lubrica (Muller). IX. Notes on the cyst, metacercaria, and infection in the ant, Formicafusca. Cornell Vet., 43: 389-410. Mapes, C.R., 1951. Studies on the biology of Dicrocoelium dendrilicum ( Rudolphi, 1819; Looss, 1899; Trematoda:Dicrocoeliidae), including its relation to the intermediate host, Cionella lubrica (Muller). I. A study of Dicrocoelium dendrilicum and Dicrocoelium infection. Cornell Vet., 41: 382-425. Massoud, J., 1981. Histopathology of liver in Iranian sheep naturally infected with Dicrocoelium dendrilicum. Ann. Trop. Med. Parasitol., 75: 293-298. McFarlane, A.S., 1958. Efficient trace labelling of proteins with iodine. Nature (London), 182: 53. Rodkey, F.L., 1965. Direct spectrum determination of albumin in human serum. Clin. Chem., 11: 478-487. Toparlak, M. and Gul, Y., 1988. Investigations on liver flukes in sheep slaughtered at local abattoirs in Van, Turkey. Vet. Fak. Dergisi Ankara Univ., 35: 269-274. Weichselbaum, T.E., 1940. Accurate and rapid method for determination of proteins in small amounts of serum and plasma. Am. J. Clin. Pathol. Tech. Sect., 10: 40-49. Wolff K., Ruosch, W. and Eckert, J., 1969. Perfusionstechnik zur Gewinnung yon Dicrocoelium dendriticum aus Schaf-und Rinderlebern. Z. Parasitenkd., 33: 85-88.