Veterinao, Parasitology, 39 (1991) 61-66 61

Elsevier Science Publishers B.V., A m s t e r d a m

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 gastrointes-
tinal helminths. In the first experiment, where groups of animals with low to high Dicrocoelium bur-
dens 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 vari-
ation 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-stand-
ing infections cause gross induration and scarring of the liver with severe cho-
langitis 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 experi-

Author to w h o m c o r r e s p o n d e n c e should be addressed.

0304-4017/91/$03.50 © 1991 - - Elsevier Science Publishers B.V.

62 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 live-
stock (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 de-
scribes 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 nat-
urally infected with D. dendriticum, were used in two experiments (Table 1 ).
All animals were removed from pasture, treated with invermectin or benzim-
idazotes 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 Radioisotopes Used

with Dicrocoelium )

Experiment 1 12 sheep 5~Cr-- red cells

previously treated with BZ ~251 - - albumin

Experiment 2 8 sheep 5 ~ C r - red cells

G r o u p X - - 4 animals ~251 - - albumin
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 63

body weights and packed red cell volumes were monitored, and samples were
obtained for serum albumin (Rodkey, 1965) and serum protein (Weichsel-
baum, 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 injec-
tion (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 multi-
channel 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, re-
spectively. 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
64 Y. THEODORIDIS ET AL

TABLE 2

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

Group Sheep Total PCV Red cell 5~Cr t~/2 Total Serum 1251 tl. 2
No. fluke volume (h) protein albumin (h)
burden (mlkg t) (gl - I ) (gl i)

A 2 0 29.5 12.00 248 74.1 34.8 328

4 100 31.0 11.00 174 80.0 32.9 260
6 60 24.5 19.00 174 84.3 31.8 288

Mean 57 28.3 14.00 199 79.5 33.2 292

B 1 840 35.0 19.60 149 70.4 41.0 314

5 680 25.5 19.70 160 94.1 34.9 422
12 300 33.5 30.80 134 85.5 34.7 405

Mean 607 31.3 23.37 148 83.3 36.9 380

C 3 1050 34.0 19.30 145 101.9 41.0 389

8 1670 23.5 12.40 139 91.5 20.4 296
9 1270 31.0 22.70 203 86.0 38.4 301

Mean 1330 29.5 18.13 162 93.1 33.3 329

D 7 3600 27.0 15.90 150 76.1 34.1 331

10 2040 27.5 17.50 163 71.6 38.7 335
11 4630 31.5 23.20 143 94.9 31.1 552

Mean 3423 28.6 18.87 152 80.9 34.6 406

PCV = packed cell volume.

TABLE 3

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

Group Sheep Total PCV Red S~Cr t]/2 Total Serum Plasma ~2Sl tE/2 SgFe l~/2
No. fluke cell (h) protein albumin volume (h) (rain)
burden volume (g1-1) (gl - I ) (mlkg 1)
(mlkg L)

X 21 2690 31.0 17.63 290 100.0 26.0 58.10 360 67.90

22 3240 31.0 17.32 339 86.0 31.0 50.70 381 105.00
24 4310 34.0 14.01 384 74.0 32.0 37.50 296 130.30
25 2430 25.5 t2.25 339 72.0 31.0 42.10 265 101.50

Mean 3163 30.4 15.30 338 83.0 30.0 47.10 326 101.18

Y 27 9 29.5 18.25 284 75.0 34.0 47.00 337 51.80

28 1 35.5 22.41 223 67.0 33.0 43.30 292 164.90
29 10 30.0 22.26 266 69.0 30.0 47.60 309 150.40
32 0 29.5 15.91 281 75.0 38.0 38.40 279 83.00

Mean 5 31.1 19.71 264 71.5 33.8 44.08 304 112.53

PCV = packed cell volume.

DICROCOELIUM DENDR177CUM INFECTION IN SHEEP 65

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 car-
rying negligible fluke burdens (Group Y). There were no significant differ-
ences 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 signifi-
cant, 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 contor-
tus (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 be-
tween 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 reticulo-
endothelial 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.
66 Y. T H E O D O R I D I S ET AL.

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