Time-dependent tegumental surface changes in
juvenile Fasciola gigantica in response to
triclabendazole treatment in goat
Author names:
P. A. Ahammed Shareef a, Gerard P. Brennan b, Paul McVeigh b, M.A. Hannan Khan a,
Russell M. Morphew c, Angela Mousley b, Nikki J. Marks b, M.K. Saifullah a, Peter M.
Brophy c, Aaron G. Maule b, S.M.A. Abidi a,*
Affiliation and address for correspondence:
a Section of Parasitology, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim
University, Aligarh 202 002, India.
b Institute for Global Food Security, School of Biological Sciences, Queen’s University
Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.
c Institute of Biological, Environmental and Rural Sciences, Aberystwyth University,
Aberystwyth, Wales, UK.
* Corresponding author
Tel.: +915712700921, Extn. 3434
Email addresses: shareefkpr@gmail.com (P.A.A. Shareef), abbasabidi92@hotmail.com
(S.M.A. Abidi)
Abstract
Triclabendazole (TCBZ), the anthelmintic drug active against both mature and immature liver
flukes, was used to investigate the effect of in vivo treatment on the tegumental surface of
juvenile Fasciola gigantica. Five goats were infected with 150 F. gigantica metacercariae
each by oral gavage. Four of them were treated with single dose of TCBZ at 10 mg/kg at four
weeks post-infection. They were euthanized at 0 (untreated), 24, 48, 72 and 96 hours post
treatment. Juvenile flukes were manually retrieved from the goat livers and processed for
scanning electron microscopy. In control flukes, the anterior region was adorned with sharply
pointed spines projecting away from the surface, while in the posterior region, spines become
shorter and narrower, loosing serration and with the appearance of distinct furrows and
papillae. The dorsal surface retained the same pattern of surface architecture similar to that of
ventral surface. Flukes obtained from 24 h post-treatment did not show any apparent change
and were still very active. However, there were limited movements and some blebbing,
swelling, deposition of tegumental secretions and some flattening displayed by the flukes of
48 h post-treatment. All the worms were found dead 72 h post-treatment and showed
advanced level of tegumental disruptions, consisting of severe distortion of spines, sloughing
off the tegument to expose the basal lamina, formation of pores and isolated patches of
lesions. By 96 h post-treatment, the disruption was extremely severe and the tegument was
completely sheared off causing deeper lesions that exposed the underlying musculature. The
disruption was more severe at posterior than anterior region and on ventral than dorsal
surface. The present study further establishes the time-course of TCBZ action in vivo with
100% efficacy against the juvenile tropical liver fluke.
Key words:
Fasciola gigantica, Triclabendazole, Scanning electron microscopy, Liver fluke, Tegumental
disruption, Goat fasciolosis
1. Introduction
Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is a serious disease
of ruminants worldwide. In some tropical countries, Fasciola infection is considered to be the
single most important helminth infection of cattle and the prevalence is as high as 30-90%
(Spithill et al., 1999). Fasciola spp. are also responsible for zoonotic infections of humans as
well. About 2.4 million people are infected with this parasite world-wide with possibly 18
million infections undiagnosed and another 180 million people living at risk of infection
(Anon, 1995; Mas-Coma et al., 1999). It has been estimated that fasciolosis causes a
substantial economic loss of at least $3.2 billion per annum to the live stock industry (Spithill
et al., 1999). The economic losses associated with fasciolosis are mainly due to decreased
weight gain and milk production and fertility as well as increased costs associated with the
need for chemotherapy and condemnation of affected livers at abattoirs (Gajewska et al.,
2005). In India, fasciolosis is a serious health problem for live-stock, with the prevalences
estimated at 43.28% (Yadav et al., 2009), therefore the economic losses associated with
fasciolosis can be predicted to be huge.
To date, no successful vaccine is commercially available to control fasciolosis, even
though many experimental vaccine trials showed varying protection to Fasciola infection
(Spithill and Dalton, 1998; McManus and Dalton, 2006; Jayaraj et al., 2009). Therefore, the
control of fasciolosis is almost exclusively dependent upon chemotherapy. Triclabendazole is
the drug of choice because of its potent flukicidal activity, covering the spectrum from early
juveniles to reproductively-mature adults (Boray et al., 1983; Rapic et al., 1988). However,
the injudicious use of can lead to TCBZ-resistance, which is a major problem with the control
of F. hepatica (Brennan et al., 2007). TCBZ is widely used for treating human Fasciola
infections as well. Possible mechanisms and pathways involved in TCBZ metabolism and
pharmacokinetic disposition in the present study have been depicted in figure 1. These
mechanisms are well studied in F. hepatica, but little is known in F. gigantica. Some studies
have reported on the action of TCBZ against both adult and juvenile flukes in vitro (Meaney
et al., 2002; Tansatit et al., 2012). In order to rule out variabilities associated with drug or ex
vivo culture, such studies must be validated in appropriate ruminant hosts. A huge gap exists
between our understanding of anthelmintic action in F. gigantica and F. hepatica. No
information is available on the effects of in vivo chemotherapy on intra-mammalian tropical
liver fluke. Therefore, in the present study, we aimed to investigate the effect of TCBZ on 4
week old juvenile F. gigantica in a goat host, using scanning electron microscopy (SEM) at
24, 48, 72 and 96 hours (h) post treatment. In contrast to tests performed in vitro, where the
flukes are exposed to a single metabolite, the pharmacokinetics of TCBZ in vivo is a
multistep metabolic pathway where the parasites are exposed to several TCBZ metabolites as
a result of hepatic metabolism of administered drug (Hennessy et al., 1987; Virkel et al.,
2006; Halferty et al., 2008; Mestorino et al., 2008). The surface tegument represents the
front-line of the host-parasite interface, and is the principal route of entry for TCBZ
metabolites into the parasite tissues (Toner et al., 2009).
2. Materials and methods
2.1. Animals and parasites
Five male goats, aged approximately 4 months old and weighing between 12 and 16
kg, were selected for the experiment, and maintained under a natural light cycle with food
and water provided ad libitum. For the study period, the animals were housed in bricked and
roofed pens with a concrete floor, and each was identified using natural markings. To confirm
no prior infection, faecal egg analyses were performed for 3 days before the commencement
of the experiment.
Each of the goats were infected with an oral gavage of 150 F. gigantica metacercariae (one
month old, stored at 4 0C in distilled water) procured from the life cycle maintained in the
Parasitology Research Laboratory at the department of Zoology, Aligarh Muslim University,
India. The experiment was approved by the animal ethical committee at the Department of
Zoology, A.M.U. Aligarh
2.2. TCBZ treatment
The goats were grouped into one control which received no TCBZ treatment and four
TCBZ treated. They were maintained on a normal feeding regime during the treatment.
Anthelmintic treatment was performed at 4 weeks post infection using an oral dose of 10
mg/kg TCBZ (“Fasinex”, 5% w/v drench). This dose has been established to be the most
effective concentration in sheep/goat against juvenile liver flukes (Boray et al., 1983; Turner
et al., 1984; Hennessy et al., 1987; Halferty et al., 2008). One animal each were sacrificed at
0, 24, 48, 72 and 96 h post treatment. Juvenile flukes were retrieved manually by crumbling
the livers in RPMI-1640 medium (Hi media Laboratories) pre-warmed to 37±0.5 0C. The
flukes were washed several times in RPMI-1640 medium and processed for scanning electron
microscopy (SEM).
2.3. Specimen preparation for SEM
Six flukes from each animal were processed for SEM analysis. Briefly, the flukes
were lightly flat fixed for 30 minutes and subsequently free fixed (in fresh fixative) for 4.5 h
at 4 0C in 4% (w/v) glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.4)
containing 3% (w/v) sucrose. Following 4 washes of 30 min each in sodium cacodylate
buffer (pH 7.4), the specimens were post fixed in 1% (w/v) osmium tetroxide for 1 h. Then
the flukes were given three washes of 10 min each in 70% (v/v) ethanol, followed by
dehydration in ascending series of ethanol. Thereafter, the specimens were dried in
hexamethyldisilazane, mounted on aluminium stubs, sputter coated with gold-palladium and
viewed in an FEI Quanta 200 SEM operating at 10 keV.
3. Results
3.1. Direct visual observation
Prior to fixing for SEM, the flukes were viewed under a stereozoom microscope. The
four week old juvenile flukes of F. gigantica recovered from goats untreated and 24 h post
treatment with 10 mg/kg-TCBZ were all very active and exhibited reddish brown gut
contents. Table I describes the visual observations of flukes recovered at each time-point.
Briefly, worms recovered from 48 h post treatment with TCBZ revealed slight reductions in
the motility and gut contents of at least 50% of worms, with the rest appeared similar to the
earlier two groups. The flukes recovered after 72 h of anthelmintic treatment were all dead
(100% efficacy), pale grey in color and exhibited no visible gut contents. The worm body
appeared elongated, tapering towards the posterior end. At 96 h post treatment, the recovered
worms were all dead, appeared pale grey in color, exhibited no gut contents and elongated
body, losing their normal texture.
3.2. Scanning electron microscopy
3.2.1. Control flukes
The tegumental surface architecture of juvenile flukes from the control group showed
normal morphology (Fig. 2). The ventral surface (Fig. 2-A) demonstrated normal rounded
oral sucker, (Fig. 2-A-1), ventral sucker (Fig. 2-A-3) and excretory pore (Fig. 2-A-5). The
anterior region, the apical cone, was adorned with sharply pointed spines projecting away
from the surface (Fig. 2-A-2). On moving toward the posterior region of the worm, the spines
become shorter and narrower, loosing serration (Fig. 2-A-4, Fig. 2-B-2), and furrows and
papillae become more prominent (Fig. 2-B-3), devoid of spines (Fig. 2-A-5, Fig. 2-B-4). The
dorsal surface (Fig. 2-B) showed the same pattern of surface architecture as that of the ventral
surface.
3.2.2. Twenty-four hours post treatment
The surface topography of juvenile flukes recovered from the goat after 24 h of
anthelmintic treatment appeared normal and apparently matched with untreated control
specimens (Fig. 3). The only notable difference observed was that the posterior region of the
flukes appeared slightly conical, known as “tapering effect”, a typical response of Fasciola to
TCBZ.
3.2.3. Forty-eight hours post treatment
Examination of the flukes following 48 h post treatment revealed variable responses,
but moderate level of damage to the tegumental surface could be seen (Fig. 4). On the ventral
side (Fig. 4-A), the morphology of the apical cone region and ventral suckers appeared
normal, however the tegumental areas bearing spines were slightly swollen at the mid-body
(Fig. 4-A-1). On moving towards posterior region, the extent of damage progresses. At the
mid region, the spines appeared deformed and partially submerged in swollen tegument (Fig.
4-A-3). The posterior mid body displayed tegumental flattening and blebbing (Fig. 4-A-4).
The posterior part showed “tapering effect”, more advanced tegumental swelling, blebbing
(Fig. 4-A-5), and aberrant meshy tegument, irregular flattened tegumental patches (arrow)
and pits formed due to their cast off (arrow head) (Fig. 4-A-6). The dorsal surface also
showed similar tegumental disruptions (Fig. 4-B). In the mid body, extensive blebbing often
burst, furrowing and swelling of the tegument which becomes flattened posteriorly with
apparent pits and patches.
3.2.4. Seventy-two hours post treatment
The flukes recovered from 72 h post treatment exhibited advanced level of disruption
as illustrated in figure 5. The examination of the ventral surface (Fig. 5-A) revealed
tegumental damage varying from partial loss to total shearing off the tegument to expose
underlying musculature (Fig. 5-A-4,6). The oral sucker appeared distorted and in the mid
body, in addition to the deformed ventral sucker, there was blebbing too. The damage was so
severe that the spines distorted completely (Fig. 5-A-2), tegument was sloughed off to expose
basal lamina and empty spine sockets, pores and isolated patches of lesions to exhibit the
parenchyma. On the dorsal side (Fig. 5-B), the tegument was completely sheared off to
expose basal lamina containing numerous pores in it (Fig. 5-B-1). The posterior body started
shrinking and when viewed at higher magnification, the damage was more severe and deeper,
and the syncytium appeared more disintegrated.
3.2.5. Ninety-six hours post treatment
The examination of the flukes revealed most advanced level of disruption and worst
damage occurred to the tegument (Fig. 6). There was complete loss of normal body texture
and the entire fluke surface had a wrinkled and corrugated appearance. Both the oral and
ventral suckers underwent extremely severe disruption which resulted in the occlusion of
these two openings (Fig. 6-A, A-1, B, B-1). The entire syncytial layer has been stripped off to
expose the basal lamina. In some specimens, the blebs and empty spine sockets (Fig. 6-B-2,
3) were retained, while all the specimens bear numerous pores in the lamina. At this time
period, there were lesions of varying sizes in the basal lamina that exposed not only the
parenchymal tissues (Fig. 6-A-2,4) but also the underlying radial and circular muscle fibres
(Fig. 6-A-3). These deformities were more prevalent at the posterior region followed by the
lateral margins.
4. Discussion
Although we have a working understanding of the impacts of TCBZ treatment on
tegumental structure in the temperate liver fluke, F. hepatica (Stitt and Fairweather, 1993,
1994; Halferty et al., 2009), the effects of TCBZ on tropical liver fluke F. gigantica,
particularly in vivo, are less well documented. In the present study we describe time
dependant progressive tegumental surface changes in 4 week old juvenile flukes of F.
gigantica in a goat model for the first time by SEM, which is a powerful technique to study
the surface topography of the tegument which has been used extensively to study
anthelmintic action in Fasciola, both in juvenile as well as adults (Tansatit et al., 2012;
Meaney et al., 2002; McConville et al., 2007; Halferty et al., 2008, 2009). Severe tegumental
disruption has been reported when adult F. gigantica was incubated in vitro with TCBZSO
(Meaney et al., 2002). Similarly, 3 week old juvenile F. gigantica obtained from
experimentally infected hamster has also been used to study the effect of anthelmintics “in
vitro” (Tansatit et al., 2012). But there is no report on the effect of TCBZ on F. gigantica in
vivo in the natural hosts. However, Halferty et al., (2008) have reported the effect of TCBZ
on 4 week old juvenile F. hepatica in sheep model. Four week post infection represents the
migratory, acute, stage of fasciolosis, exerting highest immunopathophysiological effects on
the well being of the host, and highlighting the importance of studying this specific life stage.
In the present study, the flukes from 24 h post treatment appeared unaffected, however by 48
h post treatment, the flukes showed limited damage to the tegument and by 72 h post
treatment, all the worms were dead and severely affected and, were extremely disrupted at
very advanced level by 96 h post treatment. This could be correlated with the metabolism and
pharmacokinetics of TCBZ in the treated hosts.
In cattle and goat/sheep, the rumen regulates slow release of TCBZ to the posterior
alimentary canal for their effective absorption to the blood stream (Mestorino et al., 2008).
The major part of metabolites of TCBZ is transported through the circulation by binding to
albumin which protects the drug from biotransformation or elimination resulting in its
prolonged residence in the body (Hennessy et al., 1987; Sanyal, 1995). Extremely low
concentration of circulatory TCBZ parent drug is an indication of their immediate
biotransformation into corresponding metabolites in the digestive tract or liver microsomes
and these metabolites are available in the systemic circulation concentrations that vary over
time (Hennessy et al., 1987; Kinabo and Bogan, 1988; Virkel et al., 2006; Mestorino et al.,
2008). Kinabo and Bogan (1988) reported the pharmacokinetics of TCBZ in Fasciola
infected goat. The goat plasma concentration of TCBZSO peaks (12.99 µg/ml) at 18 h postadministration, while TCBZSO2 peaks (12.11 µg/ml) after 35 h. Hydroxy-triclabendazole
(OH-TCBZ),
Hydroxy-triclabendazole
sulphoxide
(OH-TCBZSO)
and
Hydroxy-
triclabendazole sulphone (OH-TCBZSO2) form in the liver microsomes as a result of
hydroxylation of parent TCBZ, TCBZSO and TCBZSO2 respectively, and therefore these
compounds were not detected in plasma (Hennessy et al., 1987; Halferty et al., 2009).
Following intra-ruminal treatment with TCBZ, the plasma level of TCBZSO declines to ~4.5
µg/ml at 48 h, 2.2 µg/ml at 72 h and 0.9 µg/ml at 96 h respectively. Similarly, the level of
TCBZSO2 falls down to ~9 µg/ml, ~5.2 µg/ml and ~2.6 µg/ml at 48 h, 72 h and 96 h
respectively (Kinabo and Bogan, 1988).
In the present study, a progressive disruption to the tegument has been observed. All
the parent TCBZ, and their sulphoxide and sulphone metabolites induce tegumental damage,
however flukes show a region-specific susceptibility to each of the compounds (Halferty et
al., 2009). In F. hepatica, TCBZ cause highest damage to the ventral posterior midbody
followed by tail region and dorsal posterior midbody and least on oral cone. Similarly,
TCBZSO causes maximum disruption to the tail region followed by ventral posterior
midbody, dorsal posterior midbody and minimum at oral cone region. The levels of
disruptions from highest to lowest by TCBZSO2 are ventral posterior midbody, tail region,
oral cone, and dorsal posterior midbody respectively (Halferty et al., 2009). As a whole, the
disruption due to drug action in the tegument is more severe in the ventral surface and
posterior region than the dorsal surface and anterior region respectively. Similar mode of
activity observed in the present study is consistant with many previous reports both on F.
hepatica and F. gigantica (Stitt and Fairweather, 1993; Meaney et al., 2002; Halferty et al.,
2008; Tansatit et al., 2012) and also in response to other flukicides (Halferty et al., 2009).
These phenomena could be correlated with the pharmacokinetic disposition of TCBZ in
sheep/goat, where in the flukes expose to the TCBZSO2 after several hours of exposure to
TCBZSO. There by progressive disruption from posterior to anterior and ventral to dorsal
could be achieved. Susceptibility of F. gigantica to each of the TCBZ metabolites is not
known and in vitro studies have to be carried out to reveal the regional target specificity and
relative activity of individual TCBZ metabolites. The anthelmintic action could be more
severe in vivo than in vitro (Halferty et al., 2008) probably due to additive effects of the host
immune response and hostile microenvironment, as evident in the present study.
The results of the present study are comparable with that in 4 week old juveniles
(Halferty et al., 2008) and adults (Toner et al., 2010) of F. hepatica treated with TCBZ in
sheep host. The flukes recovered at 72 h post treatment in the present study were all dead;
however in F. hepatica one fluke was alive at this time point. The posterior elongation in F.
gigantica observed is consistent with both juvenile and adult F. hepatica (loc. cit.). The
surface changes in vivo in both the species include swelling, blebbing, loss of spines and
tegumental sloughing, the typical responses to benzimidazole drugs (Halferty et al., 2009). In
juvenile F. hepatica, the spines in the anterior region appeared normal after 72 h post
treatment in vivo, while in F. gigantica the spines were completely disrupted at this time
point, reflecting that the action of TCBZ in vivo is relatively severe and quicker in F.
gigantica. In adult F. hepatica, severe widespread blebbing has been observed at 48 h post
treatment in sheep host, however the blebbing is relatively mild in the juvenile flukes of both
F. hepatica and F. gigantica. In the present study, more tegumental disruption has been
observed on the lateral margins, which is in agreement with juvenile and adult F. hepatica. In
contrast to adult F. hepatica where similar extent of disruptions observed on both the dorsal
and ventral
surfaces, the juveniles of both F. gigantica and F. hepatica were displaying
more severe disruption on the ventral surface than the dorsal.
The mechanism of TCBZ action has been illustrated in figure 1. At 48 h posttreatment, there was the presence of a partial extra layer of tegumental secretion covered all
over the surface of the fluke to maintain the integrity of the apical membrane. However, since
TCBZ inhibits microtubule polymerization, the fluke cannot maintain this process
indefinitely, which ultimately lead to the disruption and consequent loss of the tegument (Stitt
and Fairweather, 1993; Meaney et al., 2004; McConville et al., 2006; Halferty et al., 2008).
Swelling and blebbing of the tegument were the initial signs of anthelmintic action, leading to
tegumental sloughing, followed by exposure and disruption of basal lamina, dislodging of
spines, generation of lesions and finally, exposure of underlying musculature. These are
likely to be anthelmint induced stress reaction where in the secretory bodies are extensively
transported to apical plasma membrane for release as an adaptive survival strategy to replace
damaged membrane and preserve the integrity of the tegument (Stitt and Fairweather, 1993;
Meaney et al., 2003; McConville et al., 2006). Binding of TCBZ to β-tubulin would prevent
the transport of secretory bodies to the tegument, due to which the apical membrane cannot
be repaired, and eventually the sloughing off the tegumental syncytium, as also observed in
the present study. Ultimately, this removes the main protective layer around the fluke, which
allows the drug to penetrate easily deeper into the tissues of the fluke. This could be the
reason for the enhanced disruption from 72 h of post treatment onwards. This phenomenon
has been observed in vitro and in vivo in juveniles and adults of both F. gigantica and F.
hepatica (Meaney et al., 2002; Halferty et al., 2008, 2009; Toner et al., 2010; Tansatit et al.,
2012). Osmotic imbalances have also been postulated to be a
possible contributing
mechanism. Benzimidazole drugs can act as an uncoupler of oxidative phosphorylation result
in decreased production of ATP which in turn would affect Na+ – K+ pump. This would lead
to the influx of Na+ and water in to the fluke and eventually swelling of the tegument, as
described in F. hepatica following nitroxynil treatment (McKinstry et al., 2003; Saowakon et
al., 2009). In the basal lamina, there were many pores which are sites for the cytoplasmic
connections to pass through the lamina from the tegumental cells (Halferty et al., 2008).
Acquiring resistance to TCBZ is an increasing problem with F. hepatica, however it is not
known/reported in F. gigantica. Since TCBZ action is similar in both the species, as
corroborated in the present study and in many previous reports, there is a great chance of the
development of resistance to TCBZ by F. gigantica, however extended studies are to be
carried out on this aspect.
It is concluded that TCBZ induces progressive tegumental disruption to four week old
migrating flukes of F. gigantica in goat. The results of the present study are consistent with
TCBZ action on F. hepatica in sheep. Oral administration of TCBZ displayed high
anthelmintic efficacy against juvenile flukes which were killed by 3 days post treatment.
Upon SEM observation, tegumental surface changes began from 48 h post treatment, starting
with blebbing and tegumental swelling, increased with time culminating in complete shearing
off the tegumental syncytium and severe lesions in the basal lamina to expose underlying
musculature. It is believed that the TCBZ action could be similar in human fasciolosis as
well. The present study established the effect of TCBZ on F. gigantica in vivo for the first
time in its natural host, unlike many previous studies conducted in vitro or in experimental
animal model systems such as rat. The time dependant tegumental disruption could be
correlated with the pharmacokinetic disposition of TCBZ and its metabolites in the goat host.
Acknowledgements
The authors wish to thank the Chairman, Department of Zoology, A.M.U., for
providing laboratory facilities. We are thankful to Dr David McCall, AFBI, Belfast, UK, for
his helping hands in SEM imaging, to Prof. R.E.B. Hanna for providing the literature and to
Mr. Rizwan Ullah, Mr. Abdur Rehman, Mr. Sarfaraz and Mr. Azam for their technical
assistance. The scanning was done while PAAS was visiting QUB for training under the
BBSRC-UK-CIDLID sponsored collaborative project, grant no. BB/H009477/1, the support
of which is gratefully acknowledged.
References
Anon, 1995. Control of foodborne trematode infections. In: World Health Organization
Technical Series No. 849, Geneva, Switzerland.
Boray, J.C., Crowfoot, P.D., Strong, M.B., Allison, J.R., Schellenbaum, M., Von Orelli, M.,
Sarasin, G., 1983. Treatment of immature and mature Fasciola hepatica infections in sheep
with triclabendazole. Vet. Rec. 113, 315–317.
Brennan, G.P., Fairweather, I., Trudgett, A., Hoey, E., McCoy, M., McConville, M., Meaney,
M., Robinson, M., McFerran, N., Ryan, L., Lanusse, C., Mottier, L., Alvarez, L., Solana, H.,
Virkel, G., Brophy, P.M., 2007. Understanding triclabendazole resistance. Exp. Mol. Pathol.
82, 104–109.
Halferty, L., Brennan, G.P., Hanna, R.E.B., Edgar, H.W., Meaney, M.M., McConville, M.,
Trudgett, A., Hoey, L., Fairweather, I., 2008. Tegumental surface changes in juvenile
Fasciola hepatica in response to treatment in vivo with triclabendazole. Vet. Parasitol. 155,
49–58.
Halferty, L., Brennan, G.P., Trudgett, A., Hoey, E.M., Fairweather, I., 2009. The relative
activity of triclabendazole metabolites against the liver fluke, Fasciola hepatica. Vet.
Parasitol. 159, 126–138.
Hennessy, D.R., Lacey, E., Steel, J.W., Prichard, R.K., 1987. The kinetics of triclabendazole
disposition in sheep. J. Vet. Pharmacol. Ther. 10, 64–72.
Jayaraj, R., Piedrafita, D., Dynon, K., Grams, R., Spithill, T.W., Smooker, P.M., 2009.
Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens. Vet.
Parasitol. 160, 230–236.
Kinabo, L.D.B., Bogan, J.A., 1988. Pharmacokinetics and efficacy of triclabendazole in goats
with induced fascioliasis disposition in sheep. J. Vet. Pharmacol. Therap. 11, 254-259.
Mas-Coma, S., Esteban, J-G., Bargues, M.D., 1999. Epidemiology of human fascioliasis: a
review and proposed new classification B. World Health Organ. 77, 340–346.
McConville, M., Brennan, G.P., McCoy, M., Castillo, R., Herna´ndez-Campos, A., Ibarra, F.,
Fairweather, I., 2007. Immature triclabendazole-resistant Fasciola hepatica: tegumental
responses to in vitro treatment with the sulphoxide metabolite of the experimental fasciolicide
compound alpha. Parasitol. Res. 100, 365–377.
McConville, M., Brennan, G.P., McCoy, M., Castillo, R., Hernández-Campos, A., Ibarra, F.,
Fairweather, I., 2006. Adult triclabendazole-resistant Fasciola hepatica: surface and
subsurface tegumental responses to in vitro treatment with the sulphoxide metabolite of the
experimental fasciolicide compound alpha. Parasitol. 133, 195–208.
McKinstry, B., Fairweather, I., Brennan, G.B., Forbes, A.B., 2003. Fasciola hepatica:
tegumental surface alterations following treatment in vivo and in vitro with notroxynil
(Trodax). Parasitol. Res. 91, 251–263.
McManus, D.P., Dalton, J.P., 2006. Vaccines against the zoonotic trematodes Schistosoma
japonicum, Fasciola hepatica and Fasciola gigantica. Parasitol. 133 (Suppl), S43–S61.
Meaney, M., Fairweather, I., Brennan, G.P., Ramasamy, P., Subramanian, P.B., 2002.
Fasciola gigantica : tegumental surface alterations following treatment in vitro with the
sulphoxide metabolite of triclabendazole. Parasitol. Res. 88, 315–325.
Meaney, M., Fairweather, I., Brennan, G.P., Forbes, A.B., 2004. Transmission electron
microscope study of the ultrastructural changes induced in the tegument and gut of Fasciola
hepatica following treatment with clorsulon. Parasitol. Res. 92, 232–241.
Meaney, M., Fairweather, I., Brennan, G.P., McDowell, L.S.L., Forbes, A.B., 2003. Fasciola
hepatica: effects of the fasciolicide clorsulon in vitro and in vivo on the tegumental surface,
and a comparison of the effects on young- and old-mature flukes. Parasitol. Res. 91, 238–
250.
Mestorino, N., Formentini, E.A., Lucas, M.F., Fernandez, C., Modamio, P., Herna´ ndez,
E.H., Errecalde, J.O., 2008. Pharmacokinetic disposition of triclabendazole in cattle and
sheep; discrimination of the order and the rate of the absorption process of its active
metabolite triclabendazole sulfoxide. Vet. Res. Commun. 32, 21–33.
Rapic, D., Dzakula, N., Sakar, D., Richard, R.J., 1988. Comparative efficacy of
triclabendazole, nitroxynil and rafoxanide against immature and mature Fasciola hepatica in
naturally infected cattle. Vet. Rec. 122, 59–62.
Sanyal, P.K., 1995. Kinetic disposition of triclabendazole in buffalo compared to cattle. J.
Vet. Pharmacol. Ther. 18, 370–374.
Saowakon, N., Tansatit, T., Wanichanon, C., Chanakul, W., Reutrakul, V., Sobhon, P., 2009.
Fasciola gigantica: Anthelmintic effect of the aqueous extract of Artocarpus lakoocha. Exp.
Parasitol. 122, 289–298.
Spithill, T.W., Dalton, J.P., 1998. Progress in Development of Liver Fluke Vaccines.
Parasitol. Today. 14, 224-228.
Spithill, T.W., Smooker, P.M., Copeman, D.B., 1999. Fasciola gigantica: epidemiology,
control, immunology and molecular biology. In: Dalton, J.P. (Ed.), Fasciolosis.CABI
Publishing, Oxon, UK, (Chapter 15), 1999, pp. 465–525.
Stitt, A.W., Fairweather, I., 1993. Fasciola hepatica: tegumental surface changes in adult and
juvenile flukes following treatment in vitro with the sulphoxide metabolite of triclabendazole
(Fasinex). Parasitol. Res. 79, 529–536.
Stitt, A.W., Fairweather, I., 1994. The effect of the sulphoxide metabolite of triclabendazole
(‘‘Fasinex’’) on the tegument of mature and immature stages of the liver fluke, Fasciola
hepatica. Parasitol. 108, 555–567.
Tansatit, T., Sahaphong, S., Riengrojpitak, S., Viyanant, V., Sobhon, P., 2012. Fasciola
gigantica: The in vitro effects of artesunate as compared to triclabendazole on the 3-weeksold juvenile. Exp. Parasitol. 131, 8–19.
Toner, E., McConvery, F., Brennan, G.P., Meaney, M., Fairweather, I., 2009. A scanning
electron microscope study on the route of entry of triclabendazole into the liver fluke,
Fasciola hepatica. Parasitol. 136, 523–535
Toner, E., Brennan, G.P., Hanna, R.E.B., Edgar, H.W., Fairweather, I., 2010. Tegumental
surface changes in adult Fasciola hepatica in response to treatment in vivo with
triclabendazole in the sheep host. Vet. Parasitol. 172, 238–248.
Virkel, G., Lifschitz, A., Sallovitz, J., Pis, A., Lanusse, C., 2006. Assessment of the main
metabolism pathways for the flukicidal compound tri-clabendazole in sheep. J. Vet.
Pharmacol. Ther. 29, 213–223.
Yadav, C.L., Rajat, G., Banerjee, P.S., Ranjan, K.R., Sanjay, Y., 2009. Epizootiology of
Fasciola gigantica infection in cattle and buffaloes in Western Uttar Pradesh, India. J. Vet.
Parasitol. 23, 135–138.
Legends to figures
Fig. 1: Mechanisms and pathways involved in triclabendazole (TCBZ) metabolism and
pharmacokinetic disposition in goat. A- Goats were previously infected with 150
metacercariae of F. gigantica and anthelmintic treatment occurred at 4 week post infection
with a single oral dose of TCBZ at 10 mg/kg. TCBZ metabolism includes ruminal (including
microflora) and hepatic biotransformations into TCBZ-sulphoxide (TCBZSO), TCBZsulphone (TCBZSO2), hydroxy-TCBZ (OH-TCBZ), hydroxy-TCBZSO (OH-TCBZSO) and
hydroxy- TCBZSO2 (OH- TCBZSO2) have been proposed (Virkelet al., 2006). Parent TCBZ
rapidly cleared from the blood by the liver (Hennessy et al., 1987) and liver microsomes
metabolize into sulpho and hydroxy metabolites (Virkelet al., 2006). Migratory juvenile
flukes are exposed to the drug in liver parenchyma (the present study) or these metabolites
are excreted into bile duct where adult flukes are bathed in bile and exposed to drug.
Eventually, these metabolites are mainly eliminated through faeces (Hennessy et al., 1987).
B- Chemical structures of TCBZ and its metabolites. C- Molecular interactions of the liver
fluke with TCBZ and its sulpho and hydroxy metabolites. The entry of TCBZ, TCBZSO,
TCBZSO2 and low amounts of OH-TCBZ, OH-TCBZSO and OH- TCBZSO2 in to the fluke
is accomplished mainly by diffusion across the tegumental syncytium rather than oral route
(Mottier et al., 2006). Within the fluke, the microsomes transform each of these entered drug
components into other metabolites, the oxidative drug metabolism (Mottier et al., 2004). Pglycoproteine (PGP) is a member of the ATP-binding cassette (ABC) transporters which
participate in ATP-dependent efflux mechanism that enable the drug to expel out from the
cells (Alvarez et al., 2007). D- All the TCBZ, TCBZSO and TCBZSO2 contribute to
anthelmintic activity and there are variations in the regional specificity in the levels of
disruption to the tegument of the fluke (Halferty et al., 2009). These TCBZ and metabolites
bind to β-tubulin which blocks the polymerization to form microtubules and consequently
disrupt microtubule based processes in the fluke (Stitt and Fairweather, 1993). This would
prevent the movement of secretory bodies from the cell body to the tegument that is vital for
the maintanace of integrity of the surface membrane which leads to severe progressive
damage to the tegument culminating in the death of the fluke (Brennan et al., 2007).
Fig. 2. Scanning electron micrographs (SEMs) of the tegumental surface of 4 week-old
control Fasciola gigantica. A- Low magnification image of the ventral surface of the fluke
showing oral sucker (i), sharply pointed spines (ii), rounded ventral sucker (iii), posterior
mid-body showing maturing spine (iv) and posterior region (v) showing excretory pore
(arrow). B- Low magnification image of the anterior dorsal surface showing spines (i),
maturing spines (ii) and on moving further to the posterior, spines disappeared completely
while the furrows and papillae are predominantly present (iii), and posterior end (iv).
Fig. 3. Scanning electron micrographs (SEMs) of the tegumental surface of 4 week-old
Fasciola gigantica 24 h post-treatment in vivo with 10 mg/kg triclabendazole, displays no
apparent visible changes to the tegument, however posterior “tapering” effect is prominent.
A- Low magnification image of the ventral surface. The oral cone region at higher
magnification showing thick spines (i), unaffected oral sucker (ii), ventral sucker (iii),
furrows and papillae (iv) at posterior mid-body and posterior cone (v) with excretory pore
(arrow). B- Low magnification image of the dorsal surface showing anterior serration with
spines (i), emerging spines (ii) and posterior cone (iii) displaying excretory pore (arrow).
Fig. 4. Scanning electron micrographs (SEMs) of the tegumental surface of 4 week-old
Fasciola gigantica 48 h post-treatment in vivo with 10 mg/kg triclabendazole. Tegumental
surface exhibits moderate level of disruption. A- Low magnification image of the ventral
surface showing anterior spines (i) appear slightly swollen, normal ventral sucker appeared
somewhat normal (ii), deformed spines partially submerged in swollen tegument (iii), blebs
(iv), swollen tegument with ruptured blebs (arrow) (v) and flattened tegumental patches
(arrow) and pits (*) formed due to their fall off. B- Low magnification image showing dorsal
surface of the fluke. Tegumental secretions paved over the tegument (i), several ruptured
blebs (iii), disruption (arrow) of tegument and disrupted bleb (white arrow) (ii) and the
surface of posterior cone (iv) exhibits disruption and flattening of pits and patches.
Fig. 5. Scanning electron micrographs (SEMs) of the tegumental surface of 4 week-old
Fasciola gigantica 72 h post-treatment in vivo with 10 mg/kg triclabendazole revealed
advanced level of disruption. A- Low magnification image of the ventral surface showing
distorted oral sucker (i), ventral sucker (iii), severely disrupted spines (ii), basal lamina has
stripped off to expose parenchyma (iv, vi), empty spine sockets and some isolated deeper
lesions (v). B- Low magnification image of the dorsal surface of the fluke. The tegument has
sloughed away completely to expose basal lamina possesses several “pores” (i), laminal
swelling and lesions (ii) and at the posterior region, the parenchyma has severely distorted
(iii). C- Anterior mid-body of another fluke showing swollen tegument (arrow) and has been
sheared off to expose basal lamina (*).
Fig. 6. Scanning electron micrographs (SEMs) of the tegumental surface of 4 week-old
Fasciola gigantica 96 h post-treatment in vivo with 10 mg/kg triclabendazole revealed most
advanced level of disruption. A- Low magnification image of the ventral surface showing
completely distorted oral sucker (i), deeper lesions to expose parenchyma (ii, iv) and worst
severely damaged posterior cone (iii) showing radial (arrow) and circular (*) muscle fibres.
B- Low magnification image of the dorso-ventrally twisted fluke showing extremely
disrupted oral cone region (i), distorted swollen basal lamina (ii) and damaged empty spine
sockets (iii) and many islets of deeper lesions can also be seen on the whole mount.
Table I. Data showing the visual observations on the motility and the gut contents of four
week old juvenile flukes of F. gigantica recovered from untreated control and TCBZ treated
goats, infected with 150 metacercarial cysts each.
Treatment (hr)
Worms recovered
Motility*
Gut contents#
Untreated
32
+++
+++
24
28
+++
+++
48
16
++
++
72
11
–
–
96
6
–
–
* Highly active (+++), moderately active (++) and immotile (–)
# Abundant (+++), less abundant (++) and absent
Fig 1
Fig 2
Fig 3
Fig 4
Fig 5
Fig 6