Parasitol Res (2002) 88: 315325

DOI 10.1007/s00436-001-0523-1

O R I GI N A L P A P E R

M. Meaney I. Fairweather G.P. Brennan

P. Ramasamy P.B. Subramanian

Fasciola gigantica : tegumental surface alterations

following treatment in vitro with the sulphoxide
metabolite of triclabendazole
Received: 27 July 2001 / Accepted: 22 August 2001 / Published online: 12 December 2001
Springer-Verlag 2001

Abstract The eect of the active sulphoxide metabolite

of the fasciolicide triclabendazole on the surface morphology of the tropical liver uke, Fasciola gigantica,
was determined in vitro by scanning electron microscopy. At a concentration of 10 lg/ml, swelling and
blebbing of the tegument was evident after only 6 h incubation. The blebbing was focussed on the spines. With
progressively longer incubation periods, blebbing on the
spines became more severe, leading to tegumental
sloughing and spine loss. Tegumental loss became more
widespread and, after incubation periods of 18 h and
24 h in vitro, perforations of the basal lamina were
evident and, in some cases, holes penetrated through the
entire uke in the tail region. The ventral surface was
consistently more severely aected than the dorsal
and similarly the posterior region of the uke was
more disrupted than the anterior region. The results
conrm the potent activity of triclabendazole against
F. gigantica.

Introduction
The giant liver uke, Fasciola gigantica, is a large
trematode parasite closely related to Fasciola hepatica. It
occurs in almost all tropical and sub-tropical regions of
the world and is the causative agent of tropical fascio-

M. Meaney I. Fairweather (&) G.P. Brennan

Fascioliasis Research Group,
The School of Biology and Biochemistry,
The Queens University of Belfast,
Belfast BT9 7BL, Northern Ireland
E-mail: i.fairweather@qub.ac.uk
Tel.: +44-28-90272298
Fax: +44-28-90236505
P. Ramasamy P.B. Subramanian
Department of Zoology,
University of Madras, Guindy Campus,
Chennai 600025, Tamil Nadu, India

losis. It is regarded as the most important single helminth parasite of ruminants in the tropics. In economic
terms, parasitism by F. gigantica costs an estimated U.S.
$3.2 billion annually (Spithill et al. 1999). This huge loss
is due to high infection rates in infected areas, the
parasites broad host range cattle, bualo, sheep,
goats, donkeys and at least 16 species of wild ruminant
and the importance of agriculture to those countries
where it occurs (Spithill et al. 1999).
While control strategies for this parasite are very
limited in most infected areas, the use of drugs to dose
infected animals is probably the most common form of
control (Fabiyi 1987; Roberts and Suhardono 1996).
Triclabendazole is the drug of choice because of its high
ecacy against both mature and immature ukes; the
latter represents the most damaging phase of the life
cycle as the immature ukes feed on the parenchymal
cells of the liver before moving into the bile duct where
they mature into adults (Misra et al. 1987; Suhardono
et al. 1991; Mahoto et al. 1994; Sanyal and Gupta 1996;
Santra et al. 1999).
The sulphoxide metabolite of triclabendazole (TCBZSX) is the active form of this drug (Hennessy et al. 1987).
Unlike other members of the benzimidazole family,
triclabendazole has no carbamate group, but instead has
a chlorinated benzene ring (Bennett and Kohler 1987).
Triclabendazole is also unusual in its spectrum of action,
being specic for F. hepatica, F. gigantica and Fascioloides magna, but having poor ecacy against nematodes,
cestodes and even other trematodes (for references, see
Fairweather and Boray 1999). This is in contrast to other
benzimidazoles, which are highly eective against most
helminths, yet have limited activity against fasciolids. The
primary action of classical benzimidazoles is widely accepted as disruption of microtubule-based processes via
binding of the drugs to the b-tubulin molecule (Lacey
1988; Nare et al. 1996). A number of possible actions have
been proposed for triclabendazole, including uncoupling
of oxidative phosphorylation in mitochondria (McCracken and Stillwell 1991; Carr et al. 1993) and disruption
of protein synthesis (Stitt et al. 1995). However,

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morphological changes in ukes following treatment with

TCBZ-SX are compatible with disruption of microtubule-based processes. For example, there was inhibition
of the transport of tegumental secretory bodies to the
apical surface of the uke, with subsequent tegumental
damage and loss (Stitt and Fairweather 1993, 1994) and
inhibition of the mitotic division of spermatogenic and
vitelline cells was also observed (Stitt and Fairweather
1992, 1996).
The tegument of the closely related F. hepatica is an
essential organ system, having crucial roles in immunoprotection, osmoregulation, nutrient uptake, secretion
and sensory perception (Fairweather et al. 1999). The
constant turnover of the surface membrane of the
tegument allows the uke to overcome the hosts
immune response. Membrane turnover is dependent on
the transport of tegumental secretory bodies from the
tegumental cells to the surface syncytium. In cells
generally, the intracellular transport of secretory vesicles
has been shown to be facilitated by networks of
microtubules (Hirokawa et al. 1998) and such a network
has been demonstrated in the tegument of F. hepatica
(Stitt et al. 1991). The organisation and functions of the
tegument in F. gigantica are likely to be similar. If, like
other benzimidazole drugs, triclabendazole acts on
microtubule-based processes, the dependence of the
tegument on these processes may make it a potential
target for the drug. The tegument is also the route
through which the drug is absorbed into the parasite.
Triclabendazole is metabolised in the liver to its active
sulphoxide form, which is then excreted in the bile and
so bathes the parasites tegument.
The present study was carried out by means of
scanning electron microscopy (SEM) to assess whether
TCBZ-SX has any eect on the surface architecture of
F. gigantica following incubation in vitro. This is the rst
morphological study of drug eects on F. gigantica.

Materials and methods

Adult F. gigantica were recovered from the bile ducts of bualo
killed in the Peramber slaughterhouse in Chennai (Madras), South
India and transported to the laboratory in warm (37C) HedonFleig saline. Flukes were washed in several changes of warm (37C)
NCTC 135 culture medium containing antibiotics (penicillin,
50 IU/ml; streptomycin, 50 lg/ml) and then incubated whole for
either 6 h, 12 h, 18 h or 24 h in fresh NCTC 135 culture medium
containing TCBZ-SX at a concentration of 10 lg/ml. Eight ukes
were incubated per time period. The concentration of TCBZ-SX
was chosen to correspond to maximum blood levels in vivo:
9.64 lg/ml in cattle following a therapeutic dose of 12 mg/kg
(Sanyal 1995); 5.7 lg/ml in bualo following a therapeutic dose of
24 mg/kg (Sanyal and Gupta 1996). NCTC 135 and antibiotics
were obtained from Flow Laboratories, Irvine, Ayrshire, UK The
drug was initially prepared as a stock solution in dimethyl sulphoxide (DMSO) and added to the culture medium to give a maximum solvent concentration of 0.1% (v/v). Control ukes were
incubated for 6 h, 12 h, 18 h and 24 h in NCTC 135 culture
medium containing 0.1% (v/v) DMSO. Immediately after washing,
0 h controls were xed . Following incubation, ukes were lightly
at-xed for 15 min and then xed overnight at 4C in a 3:1 mix of

4% (w/v) aqueous glutaraldehyde and 1% aqueous osmium

tetroxide. Specimens were washed several times in NCTC 135,
dehydrated through an ascending series of acetones, dried in an
Emscope 750 critical-point drier, mounted on aluminium stubs and
sputter coated with gold-palladium. Specimens were viewed in an
ISI ABT-55 SEM operating at 10 keV.
All experiments comply with the current laws of the country in
which they were carried out.

Results
Normal morphology
The surface morphology of F. gigantica was recently
described in detail by Dangprasert et al. (2001) and so
will not be repeated here. However, a brief description
of some of the important features is necessary in order
to appreciate the changes resulting from treatment with
TCBZ-SX. Adult F. gigantica are leaf-like in shape and
approximately 40 mm in length, with tapered anterior
and posterior ends. At the anterior end of the uke is a
prominent apical cone with the oral sucker at its tip
and is bounded posteriorly by the ventral sucker. The
gonopore is situated two thirds of the way back
between the oral and ventral sucker (Fig. 1). When the
uke was examined by SEM, the entire surface of the
tegument, with the exception of the rims of both
suckers, was seen to be covered in spines (Figs. 1, 2).
The size and shape of these spines were dependent on
the area of the uke on which they were present. Spines
were larger on the anterior and anterior mid-body
regions than on the posterior mid-body and tail
regions. The spines from the anterior regions also
diered in shape, having nger-like protrusions at their
tips (Fig. 3); the protrusions were either greatly reduced
or absent from the spines which were observed in the
posterior mid-body and tail regions (Fig. 4). Moving
posteriorly along the body of the uke, the number of
spines present also decreased (Fig. 5). At high magnication, the tegumental surface of the uke between
the spines was seen to be made up of a meshwork of
tiny ridges and pits (Fig. 4); this meshwork was present
on all areas of the uke, except the rims of both
suckers.
Six-hour drug treatment
After 6 h incubation in vitro in TCBZ-SX (10 lg/ml),
the apical cone region showed relatively little disruption,
apart from some swelling of the tegument on the dorsal
surface (Fig. 6). Due to the swelling of the tegument
around them, the spines did not protrude as clearly from
the surface. In the mid-body region, the most severe
swelling was observed on the ventral surface directly
behind the ventral sucker. Swelling was also present
towards the lateral margins of the uke on both the
dorsal and the ventral surfaces, although the swelling
was more prominent on the ventral surface (Figs. 7, 8).

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Figs. 15 Scanning electron
micrographs of control adult
Fasciola gigantica
Fig. 1 Ventral surface of the
apical cone showing the oral
sucker (OS), ventral sucker
(VS) and gonopore (g). The
surface of the uke is covered with spines. Bar 400 lm
Fig. 2 Dorsal surface of the
apical cone region with the oral
sucker (OS) at its anterior end.
Bar 300 lm
Fig. 3 Spine (S) from the ventral surface of the apical cone
region showing the numerous
large nger-like projections
(arrow) at its tip. Bar 3 lm
Fig. 4 Spine (S) from the
dorsal surface of the anterior
mid-body region of control
adult F. gigantica showing fewer protrusions (arrow) at its tip
than seen on spines from the
anterior region. The surface of
the tegument covering the
spines takes the form of a
meshwork of ridges and pits (r).
Bar 2 lm
Fig. 5 Dorsal surface of the tail
region of control adult
F. gigantica showing a lower
density of spines than seen in
the anterior half of the uke.
Bar 100 lm

The spines appeared to be submerged by the swollen

tegument around them (Fig. 8).
In the mid-body region of the ukes, disruption in the
form of blebbing was also observed and was considerably more severe on the ventral surface (Fig. 9), where it
appeared to be concentrated o the midline of the uke
towards the lateral margins and was especially severe in
the posterior mid-body region. On the dorsal surface,

blebbing was less severe and was not concentrated in any

particular area to the same extent as that seen on the
ventral surface. Blebbing on the dorsal surface also
became more severe towards the posterior mid-body
region of the uke. The blebs appeared to form initially
on the tips and upper surfaces of the spines and then
spread to the areas between the spines as the blebbing
became more extensive (Fig. 9). Often in these areas of

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severe blebbing, the tegument covering the tips of the

spines appeared to have ruptured and the tegument was
beginning to slough away. Another prominent feature of
the 6 h-treated material was the disruption to the upper
surfaces of the spines, especially in the posterior midbody and tail regions of the uke (Fig. 10). This took the
form of a build-up of rubble on the upper surfaces of
the spines and, on closer examination, seemed to be the

beginnings of removal of the tegumental membrane

(Fig. 10). In the tail region of the uke, some blebbing
was present, to a lesser extent than that seen in the midbody region, but what was apparent in this region was
that a large number of spines had lost the tegument from
their tips. This appeared to aect the spines on the
ventral surface more severely than those on the dorsal
surface (Fig. 11).

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b

Figs. 611 Scanning electron micrographs of adult F. gigantica

which had been treated with TCBZ-SX (10 lg/ml) for 6 h in vitro
Fig. 6 SEM showing localised swelling on the dorsal surface of
the apical cone with spines (arrows) almost submerged by the
surrounding tegument. Bar 100 lm
Fig. 7 SEM showing swelling of the tegument (arrows) around the
spines (S) on the dorsal surface of the anterior mid-body region. Bar
20 lm
Fig. 8 SEM showing swelling of the tegument (arrows) around the
spines (S) on the ventral surface of the posterior mid-body region.
Bar 20 lm
Fig. 9 SEM showing blebbing (b) on the tips of the spines from the
lateral margin of the anterior mid-body region ventral surface and
also swelling of the tegument around the spines (arrow). Bar 10 lm.
The inset shows a higher power SEM of a spine from the same
region. Bar 5 lm
Fig. 10 SEM showing spines (S) from the ventral surface of the
posterior mid-body region, with disruption to the tegument on the
upper surfaces and tips of the spines; this disruption appears as a
build up of rubble on the surface of the tegument (arrows). Bar
20 lm. The inset shows a spine at higher magnication: the
tegument at the tip and on the upper surface of the spine can be
seen to be severely disrupted. Bar 5 lm
Fig. 11 SEM of spines on the ventral surface in the tail region of
the uke, showing disruption in the form of sloughing of the
tegument from the tips and upper surfaces of the spines (arrows).
Bar 20 lm

Twelve-hour drug treatment

After 12 h incubation in TCBZ-SX, tegumental disruption in the apical cone region became more pronounced
and the ventral surface was more severely aected than
the dorsal surface. Disruption to the spines, in the form
of tegumental sloughing at the tips, was more widespread
in the apical cone region than that seen in the 6 h-treated
material. The tegumental swelling which was seen in the
previous time period was more pronounced on both
dorsal and ventral surfaces. Tegumental disruption, in
the form of sloughing of the tegument around the spine
tips and in some cases complete spine loss from the
tegument, was only extensive on the ventral surface of the
apical cone, although in some small isolated areas on
the dorsal surface of the apical cone, spines had been
removed from the tegument. Immediately behind the
ventral sucker, in the anterior mid-body region, severe
tegumental sloughing was evident and, in some cases, the
tegument was completely removed, exposing the basal
lamina and empty spine sockets (Fig. 12). In specimens
where the syncytium in this area was still intact, it was
severely ssured and pitted and the majority of the spines
had been removed from the tegument. In most of the
specimens examined, the disruption continued down the
centre of the body well into the anterior mid-body region.
There was also extensive and severe swelling in the midbody region of the 12 h-treated samples towards the
lateral margins of the ukes on both the dorsal and
ventral surfaces (Figs. 13, 14). Blebbing was also extensive in the 12 h-treated specimens and was considerably
more severe on the ventral surface (Fig. 14), where it still

appeared to be concentrated towards the lateral margins.

The blebbing observed on the dorsal surface was more
severe than that observed in 6 h-treated ukes (Fig. 13).
In the mid-body region, a larger number of spines had
damage to the tegument around their tips than was observed in the apical cone region or any area of the 6 htreated specimens. This disruption was especially severe
in the posterior mid-body region. The tail region of the
12 h drug-treated ukes was most severely disrupted,
with a number of areas having lost spines. Fissuring and
pitting of the tegument were more extensive and patches
of tegument had completely sloughed o (Fig. 15). This
damage was generally more prominent o the mid-line of
the body. Such tegumental damage was also present on
the dorsal surface, but to a lesser degree. On both the
dorsal and ventral surfaces in the tail region, blebbing
was less severe than that seen in the mid-body region.
Eighteen-hour drug treatment
After 18 h treatment in TCBZ-SX, severe disruption was
evident in all regions of the uke. In the apical cone
region, spines were completely removed from most areas
and the vast majority of the remaining spines had severe
damage to the tegument covering their tips. The tegumental surface between the spines on the apical cone was
ssured and pitted and, in some areas of the ventral
surface, patches of tegument had been completely
removed, exposing the basal lamina (Fig. 16). On the
dorsal surface of the apical cone, ssuring and pitting of
the tegument was also present, but was less extensive,
and in the majority of specimens examined no tegument
removal had occurred. In the mid-body region, severe
damage was apparent on both the ventral and dorsal
surfaces. Large areas of tegument were completely
removed, exposing the basal lamina with empty spine
sockets (Fig. 17). In areas where the tegument remained,
the surface was ssured and pitted and devoid of spines
(Fig. 18). This disruption was more severe in the
posterior than the anterior mid-body region (Fig. 19). In
the tail region of the uke, even greater disruption of the
tegument had occurred. On the ventral surface of the
uke in this region, small holes had begun to form which
penetrated the basal lamina in areas where tegument had
been removed (Fig. 20). While the disruption to the
dorsal surface was severe, it was not as extensive as that
seen on the ventral surface and no holes were observed
in the basal lamina.
Twenty-four-hour drug treatment
After 24 h incubation in TCBZ-SX, the tegument was
almost entirely removed from the ventral surface of
the apical cone region, apart from a small area around
the gonopore region and immediately anterior to the
ventral sucker (Fig. 21). Although the disruption to the
dorsal surface was severe, with almost complete loss of

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Figs. 1215 Scanning electron
micrographs of adult F. gigantica which have been treated
with TCBZ-SX (10 lg/ml) for
12 h in vitro
Fig. 12 SEM of the area immediately behind the ventral
sucker showing sloughing of the
tegumental syncytium. Empty
spine sockets (SS) can also be
seen in the basal lamina. Bar
=200 lm
Fig. 13 SEM of the dorsal
surface in the lateral region of
the anterior mid-body, showing
a carpet of small blebs
(b) between the spines (S).
Bar 10 lm
Fig. 14 SEM of the ventral
surface along the lateral margin in the anterior mid-body
region showing an area extensively carpeted with large blebs
(b) and also severe swelling
between the spines (arrows).
Bar 10 lm. The inset shows
blebbing at higher magnication. Bar 10 lm
Fig. 15 SEM of the ventral
surface in the tail region of the
uke showing a large area of
exposed basal lamina (bl) with
empty spine sockets (SS). The
remaining tegument is severely
ssured and pitted (arrows)
and has lost the majority of its
spines. Bar 100 lm

spines and severe ssuring and pitting of the tegument,

in most samples only small isolated areas of the syncytium were completely sloughed away to expose the basal
lamina (Fig. 22). In the mid-body region, tegument loss
was also very extensive and in almost all specimens
examined what tegument remained was ssured, pitted
and sloughing away (Fig. 23). This was true of both the
ventral and the dorsal surfaces, although the sloughing

was more severe in the posterior mid-body than the

anterior mid-body region. In the tail region of the uke,
very little tegument remained on the ventral surface
(Fig. 24). The holes described in the previous incubation
period were now widespread and, in some specimens,
penetrated through the entire uke (Fig. 25). On the
dorsal surface, some tegument was present in most
specimens, and no holes were seen, except for those

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Figs. 1620 Scanning electron micrographs of adult
F. gigantica which have been
treated with TCBZ-SX
(10 lg/ml) for 18 h in vitro
Fig. 16 SEM of the ventral
surface of the apical cone
showing exposed basal lamina (bl) around the ventral
sucker and gonopore.
Fissuring and pitting
(arrows) of the remaining
tegument and empty spine
sockets (SS) are also evident.
Bar 100 lm
Fig. 17 SEM of the dorsal
surface along the lateral margin in the anterior mid-body
region showing ssuring and
pitting (arrows) of the tegument. Almost all spines have
been removed, leaving empty
spine sockets (SS). Bar 100 lm
Fig. 18 SEM of the dorsal surface in the anterior mid-body
region showing areas of tegumental sloughing and spine
loss. Bar 20 lm
Fig. 19 SEM of the ventral
surface in the posterior midbody region showing a hole
(h) through the basal lamina.
The tegument in the area
around the hole has sloughed
o, revealing empty spine
sockets. Bar 10 lm
Fig. 20 SEM of the ventral
surface in the tail region showing a series of holes (h) through
the basal lamina; they are
surrounded by an area of
exposed basal lamina (bl) from
which the tegumental syncytium has been removed. Empty
spine sockets are evident. Bar
100 lm

which penetrated the entire body and thus had no

obvious point of origin.
Control specimens
No appreciable dierence was observed between any of
the control specimens.

Discussion
The use of SEM in this study gave an overview of the
normal surface architecture of the tegument of
F. gigantica, thus establishing any regional variations.
This then enabled any changes due to drug action to be
properly evaluated. In the present study, regional

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dierences in response to drug action were observed,

with the ventral surface being more severely disrupted
than the dorsal and the tail region of the uke more
disrupted than the anterior.
SEM has previously been used in a number of other
studies pertaining to drug treatment of the closely
related trematode, F. hepatica (e.g. Anderson and

Fairweather 1988; Skuce and Fairweather 1990; Stitt

and Fairweather 1993). From the present study, it is
evident that TCBZ-SX has a rapid and severe eect on
F. gigantica, with widespread disruption to its tegument
present after only 6 h incubation in the drug. This
disruption became noticeably more severe after longer
incubation periods and appeared to follow a specic

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b

Figs. 2125 Scanning electron micrographs of adult F. gigantica

which have been treated with TCBZ-SX (10 lg/ml) for 24 h in vitro
Fig. 21 SEM of the ventral surface of the apical cone. Only small
areas of tegumental syncytium remain; the rest of the tegument in
this region has been removed (arrows). Bar 200 lm
Fig. 22 SEM of the dorsal surface of the apical cone showing
empty spine sockets (SS) where spines have been lost from the
tegument. Patches of exposed basal lamina (bl) can also be seen
where the tegumental syncytium has been sloughed away. Bar
300 lm
Fig. 23 SEM of the ventral surface in the posterior mid-body
region showing a large area of exposed basal lamina (bl) with
empty spine sockets (SS). Bar 200 lm
Fig. 24 SEM of the ventral surface in the tail region showing large
areas of exposed basal lamina (bl) where the tegumental syncytium
has been sloughed away. Bar 500 lm
Fig. 25 SEM of the ventral surface in the posterior region showing
a large hole (H) which has penetrated through the entire body of
the uke and a smaller holes (h) which has penetrated through the
basal lamina. Bar 200 lm

course, starting with swelling of the tegument and

blebbing at the tips of the spines. After longer incubation periods, the tegument at the tips of the spines was
the rst area to be sloughed away. When the disruption
of the tegument became more severe, spines detached
from the syncytium, then more widespread sloughing
occurred. Finally, following removal of large areas of
the tegumental syncytium, holes began to form in the
basal lamina. In the longest incubation period (24 h), the
holes had increased in size and, in some instances, even
penetrated through the entire body of the uke.
Surface blebbing is a common feature of drug-treated
parasites and has been described for F. hepatica
following incubation with a number of anthelmintics
(Fairweather et al. 1987; Anderson and Fairweather
1988; Stitt and Fairweather 1993). This form of
disruption has also been observed in other atworm
parasites (Borgers et al. 1975; Bricker et al. 1983) and is
believed to be a calcium-dependent process (Bricker et al.
1983; Xiao et al. 1984). In F. hepatica, it has been suggested that the blebbing occurs as a result of increased
eorts on the part of the parasite to shed and replace
outer tegumental membrane damaged by drug action
(Stitt and Fairweather 1993). It may be signicant, then,
that the process of blebbing appears to start at the tips
of the spines. It has been shown that tubulin, the main
component of microtubules, is present in the tegumental
syncytium, stretching vertically from the base to the
apical surface and horizontally beneath the apex (Stitt
et al. 1991). The exception to this is around the spines,
where microtubules are only present as a horizontal
band beneath the apex. Since transport of tegumental
secretory bodies is dependent on microtubules and the
tegumental membrane around the spines in drugaected ukes is not being provided with a direct supply
of replacement membrane components, this area may
represent a weak point in the tegumental membrane. If
the primary action of triclabendazole, like other benz-

imidazoles, is in fact disruption of microtubule-based

processes, it would seem reasonable to suggest that an
area heavily dependent on ecient and properly
organised transport of secretory bodies, namely the
spine tips, would show the greatest disruption. This is
also the area from which the tegument is rst lost and,
when tegumental disruption becomes more severe,
spines are completely lost from the syncytium, the empty
spine sockets allowing further penetration of the drug
into the syncytium. It may be that as the incubation time
increases, transport is eventually disrupted throughout
the syncytium and so plasma membrane cannot be
replaced quickly enough to prevent ssuring and pitting
of the tegument, leading to the eventual complete loss of
the syncytium. The basal lamina is exposed and then
breached, creating holes and enabling access of the drug
to the internal tissues, causing further and greater
damage to the uke.
Regional dierences in the disruption caused by
TCBZ-SX can clearly be seen, especially after the longer
incubation periods. The dorsal surface appears less
severely aected than the ventral and the anterior region
of the uke less severely aected than the posterior.
These regional dierences are similar to those described
previously after treatment of F. hepatica with TCBZ-SX
(Stitt and Fairweather 1993), although in the latter study
the dorsal-ventral regional dierence was only observed
in juvenile ukes (3 weeks old). Dorsal/ventral dierences in drug response have been described in studies
involving bithionol and diamphenethide; in these studies, the dorsal surface was the more severely aected
(Dawes 1966; Anderson and Fairweather 1988). Greater
damage to the tail region of F. hepatica resulted from
treatment with bithionol, closantel, hexachlorophene
and tetrachlorodiuoroethane (Boray and Pearson 1960;
Dawes 1966; Guselnikova 1974; Verheyen et al. 1980).
For the last-mentioned drug, a severe ascending necrosis
was described, with only the oral cone and suckers still
active, while the rest of the body was either necrotic or
missing altogether (Boray and Pearson 1960). Similar
serious damage to the posterior region of the uke has
been observed in mebendazole-treated ukes in vivo
(Verheyen et al. 1982). In contrast to the posterioranterior spread of damage just described, diamphenethide and clorsulon induce an anterior-posterior spread
of damage in F. hepatica (Anderson and Fairweather
1988; Fairweather and Boray 1999). It has been
suggested that the regional dierences are due to
developmental changes in tegumental architecture and
ion pump activity (Fairweather et al. 1986; Anderson
and Fairweather 1988).
The disruption caused by TCBZ-SX to the tegument
of F. gigantica is very similar to that described for
F. hepatica following treatment with this drug (Stitt and
Fairweather 1993). It is also similar to that observed in
F. hepatica and other atworm parasites following
exposure to mebendazole, another benzimidazole compound (Borgers et al. 1975; Verheyen et al. 1982). This
might suggest that, while TCBZ may not bind to the

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b-tubulin molecule at the same binding site as other

benzimidazoles (Lacey 1988; Nare et al. 1996;
Fairweather and Boray 1999), its eects may be caused
by disruption of the same target, perhaps via binding to
an alternative site on the b-tubulin molecule.
What is evident from this study is that TCBZ-SX has
a potent, rapid and progressive eect on the tegument of
F. gigantica. The surface changes induced by this drug in
vitro may serve to illustrate why it is so eective against
F. gigantica in vivo where, if the tegument of the parasite
was disrupted, the surfactant properties of bile would
also have an eect on the parasite. Any drug which has
the potential to target the tegument of a parasite in such
a destructive fashion will have good ecacy against that
parasite due to the essential roles of the tegument in
immunoprotection, osmoregulation, nutrient uptake,
sensory perception and secretion. The ability of TCBZSX, when used at therapeutic levels as it was in these
experiments, to cause almost complete removal of the
tegument of F. gigantica means that it eectively
destroys the parasites rst and main line of defence and
also allows the drug potential access to other, internal
tissues of the uke, which will lead to more widespread
damage.

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