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Original article

107

Effect of nitroxynil (fasciolid) on adult Fasciola gigantica

and Fasciola hepatica in infected cows
Eman K. Omran, Noha S. Ahmad
Department of Medical Parasitology, Faculty of
Medicine, Sohag University, Sohag, Egypt
Correspondence to Eman K. Omran, PhD,
Department of Medical Parasitology, Faculty of
Medicine, Sohag University, Sohag 2630, Egypt
Tel: +20 100 307 7912/20 115 510 5606;
fax: 002 093 4602963
e-mail: moabdallah.1998@hotmail.com
Received 12 December 2014
Accepted 25 May 2015
Parasitologists United Journal
2015 8:107114

Background
Drug resistance to treatment of fascioliasis with triclabendazole (TCBZ) has emerged in Sohag
Governorate, Egypt. Nitroxynil belongs to the halogenated phenol group of fasciolicides. It is
highly active against adult liver flukes. A nitroxynil metabolite is produced in the liver parenchyma
adding to its flukicidal activity and augmenting its efficacy against late immature flukes that
migrate through the liver tissues. Treatment with nitroxynil may be an effective replacement
for therapy with TCBZ in cases of resistance.

Objective
The aim of this study was to evaluate the efficacy of nitroxynil in the treatment of fascioliasis
by assessing its effect on teguments and durability of adult Fasciola gigantica and Fasciola
hepatica worms.

Materials and methods

Infected cows were selected on the basis of clinical signs, and infection was confirmed by
detection of Fasciola eggs in their stools. Nitroxynil was administered as recommended in
two doses 15 days apart, and the animals were slaughtered 15 days after treatment. Fasciola
worms collected from the bile ducts were identified and prepared for electron microscopy.
Tegument changes were examined with scanning electron microscopy.

Results
The removed adult flukes of both species were moving sluggishly and appeared pale with no
evidence of gut content. Scanning electron microscopy examination of these flukes revealed
evidence of swelling of the tegument that showed regional variation in its severity. Loss of
spines was also observed.

Conclusion
The present study demonstrated the flukicidal properties of nitroxynil, proving that the tegument
is an important target for its action. Disruption of the flukes main line of defense allowed the
drug access to other internal tissues, leading to more widespread damage. Nitroxynil may be
successfully used for treatment in case of resistance to TCBZ.

Keywords:
adult Fasciola, in vivo treatment, flukicides, nitroxynil (flukicide), scanning electron microscope
Parasitologists United Journal 8:107114
2015 Egyptian Parasitologists United Society
1687-7942

Introduction
Fascioliasis is one of the most important parasitic
diseases in domestic ruminants throughout the world
caused by the trematodes Fasciola gigantica and Fasciola
hepatica. Both flukes are economically significant
parasites in livestock constituting one of the important
causes of zoonotic infections. In Egypt, fascioliasis
has been recorded as an important clinical problem,
particularly among school-aged children living in rural
areas of the Nile Delta [1,2]. Animal as well as human
fascioliasis is a growing problem, as it has been recorded
in almost all governorates, especially those of the Nile
Delta in Lower Egypt [3]. A large variety of animals,
such as sheep, goats, cattle, buffalo, horses, donkeys,
camels, and rabbits, showed Fasciola infection rates
that may reach 90% in some areas [4]. Among these
animal species, fascioliasis is highly endemic in sheep
as indicated by macroscopic detection of liver flukes in
slaughtered sheep during abattoir surveys (20.6%) [5].

In another report from North Sinai, 12.7% of Fasciola

spp. were recorded in sheep and goats by microscopic
detection of eggs [6]. In the same year, infection was
found to occur mostly in sheep (17.84%), followed
by cows (12.31%), buffaloes (9.73%), and lastly goats
(5.40%) [7]. Reported prevalence rates for Fasciola
infections have reduced in recent years due to control
measures enforced in the Egyptian governorates,
including triclabendazole (TCBZ) administration [2].
In the absence of any effective vaccines, the principal
method of control remains to be the use of drugs.
Treatment with TCBZ, which is a member of the

This is an open access arcle distributed under the terms of the Creave
Commons Aribuon-NonCommercial-ShareAlike 3.0 License, which allows
others to remix, tweak, and build upon the work non-commercially, as
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2015 Parasitologists United Journal | Published by Wolters Kluwer - Medknow

DOI: 10.4103/1687-7942.175008

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benzimidazole family of anthelminthics, is the main

therapy for control of fascioliasis morbidity [8].
It works by preventing the polymerization of the
tubulin molecule into the cytoskeletal microtubule
structures[9,10] and is particularly active against both
juvenile and adult flukes [11]. Likewise, closantel,
nitroxynil, rafoxanide, and TCBZ are among the
few flukicides that are active against both mature
and immature liver flukes. Other flukicides such as
albendazole, clorsulon, and oxyclozanide are active
against adult liver fluke only [12].

of the gastrodermis also increased in severity over time,

although it was not as severely affected as the tegument.
They added that, notably, the recovered flukes displayed
disruption of the subtegumental muscle bundles that
appeared widely separated, as well as progressive loss
of fibers that rendered the muscle blocks almost empty
and barely recognizable [32]. It was noted that, on
lengthening the period of treatment with 40 mg/kg
nitroxynil, the activity of the adult fluke was affected
up to immobility [33]. The earliest tegumental changes
were recorded after 24 h from onset of treatment.

However, resistance of F. hepatica to TCBZ has been

registered since 1990 [13] and has since been reported
in Australia [14], Ireland [15], the Netherlands [16],
and southern Wales [17]. It is now known to exist in
a number of European countries as well [9,18], and
in Egypt [19]. A number of fasciolicides (including
nitroxynil) have been evaluated against TCBZ-resistant
flukes [20].

Most recently, in 2015, Junquera [34] stated that, in

contrast to other anthelminthics (e.g. imidazothiazoles,
benzimidazoles, and tetrahydropyrimidines), nitroxynil
has a residual effect i.e., it not only kills the parasites
present in the host at the time of treatment, but
also protects against reinfestation for a period of
up to several weeks, depending on the dose and the
specific parasite. This further collaborates the former
declaration that nitroxynil can be used as an alternative
to TCBZ in therapy of resistant cases [35].

Nitroxynil (4-hydroxy-3-iodo-5-nitrobenzonitrile),
which is a halogenated phenol [21,22], acts by stopping
oxidative phosphorylation in the cell mitochondria.
This disturbs the production of ATP, thus impairing
motility of the parasites [23,24]. Nitroxynil binds
very strongly (9798%) to plasma proteins [25],
indicating that oral ingestion is the most likely route.
Its nematocidal activity against adult and larval stages
of Haemonchus contortus in sheep and Haemonchus
placei, Oesophagostomum radiatum, and Bunostomum
phlebotomum in cattle was previously recorded [26].
It was reportedly active against TCBZ-resistant
F. hepatica [27] and was found synergistically active
when therapy was tested in combination with clorsulon
or closantel in the treatment of salicylanilide-resistant
flukes, even when administered in lower than
recommended doses [28]. Therapy with nitroxynil
gave 100% efficacy against Fasciola spp. in all posttreatment observations [29,30]. Efficacies around
100% have also been described under field [31] and
experimental [27] conditions.
The integrity of the surface plasma membrane and
the tegumental syncytium is essential as a first line
of defense for flukes. The tegument is the primary
drug target immediately exposed to anthelminthics.
In 2010, Toner et al. [32] observed that, due to the
severity of changes produced by TCBZ treatment,
the flukes become eliminated from the host through the
gall bladder and that their migration corresponds with
the degenerative changes. In their detailed report, they
described progressive disruption of the tegumental
system and musculature, which became severe with time
of exposure, up to complete sloughing of the tegument
and degeneration of the underlying tissues. Disruption

Scanning electron microscopy (SEM) has been

successfully used for evaluating anthelminthic effect
on changes of tegumental surface of flukes. SEM
allows scanning of the whole-body surface before
examination with transmission electron microscope of
internal structure changes [33], revealing the location
and possible variation of any disruption caused by a
drug [36]. Therefore, the aim of the present study was
to evaluate the efficacy of nitroxynil for the treatment
F. gigantica and F. hepatica in naturally infected cows,
by noting its action on the tegument and durability
after treatment.

Materials and methods

Type of study

This is a clinical veterinary trial.

Animal source of flukes

Infected cows were selected from Tahta Veterinary

Clinic on the basis of clinical signs such as foulsmelling watery diarrhea, anorexia, and hypothermia
with abrupt decrease in milk yield. Natural infection
was determined by detection of Fasciola eggs in the
cows stools using direct smear and sedimentation
methods.
Drug

Nitroxynil 1 ml/25 kg body weight was given orally

to two naturally infected cows according to the
instructions of Chemical Industries Development

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Effect of nitroxynil on adult Fasciola Omran and Ahmad

under license of Pharmachim Bulgaria. Two doses

were given 15 days apart in an attempt to avoid any
side effects.
Parasite collection

Flukes identified as adult F. gigantica and F. hepatica

were recovered from the bile ducts of slaughtered cows
15 days after treatment and washed in several changes
of warm (37C) sterile physiological saline to remove
any attached particles.
Scanning electron microscopy studies

Preparation of samples for SEM was carried out

as described [37]. Washed flukes were fixed in 5%
glutaraldehyde for 2472 h and washed four times
in sodium cacodylate buffer (pH 7.3) for about 15
min. Each postfixation was carried out by adding 1%
osmium tetroxide for 2 h and then the samples were
washed three times in the same buffer. Dehydration
was carried out in ascending concentrations of ethanol
(30%, 50%, 70%, and 90%), each for 30 min, and in
double changes of absolute ethanol for 2448 h. The
samples were incubated at 2025C on double-sided
carbon scotch tape and coated with gold. The samples
were prepared, examined, and photographed at the
SEM Unit, Assuit University.

109

tegumental surfaces, with regional variation in the

severity of the swelling. On the ventral surfaces of the
flukes, moderate swelling of the teguments was observed
around the oral and ventral suckers (Figs. 1 and 2). In
the central regions of the oral cones, the teguments
were severely swollen. The swelling was less severe along
the lateral margins of the oral cones. At the same time,
the spines were barely visible (Figs. 3 and 4). In the
anterior mid-body regions, directly behind the ventral
suckers, no spines were visible as they had become
completely submerged by the externally swollen
teguments (Figs. 5 and 6). The mid-body regions
displayed widespread swelling and extensive furrowing
of the teguments. The spines were almost submerged
by the surrounding swollen teguments (Figs. 7 and 8).
The dorsal surfaces were similarly acutely affected as
the ventral surfaces. The spines appeared to be flaking
off, and there were multiple pores and furrowing of the
teguments (Figs.9 and 10).

Results
Flukes recovered from the bile ducts of cows 15 days
after treatment were moving sluggishly and appeared
pale with no evidence of gut contents. The SEM
examination revealed evidence of swelling of the

Discussion
Treatment of animal fascioliasis is imperative for the
management of economic losses and for avoiding
zoonotic infections. Cows and sheep pose as the
main hosts for both Fasciola spp. and are responsible
for passing the infection to man [38]. The effective
control of these infections with anthelminthic
drugs is therefore vital for both animal welfare and
productivity [21,39,40]. The drug of choice in the
treatment of fascioliasis has been TCBZ, until the flukes
started to exhibit their resistance to it [13]. Resistance
of liver flukes to treatment with nitroxynil has not
reached the scale experienced with nematodes, but it

Figure 1

Figure 2

SEM of F. gigantica after treatment in vivo with Nitroxynil 1 cc/25

kg BW. Ventral Surface: The apical cone and the anterior mid-body
regions revealed tegumental swelling with regional variations in
severity surrounding the oral and ventral suckers (arrow).

SEM of F. gigantica after treatment in vivo with Nitroxynil 1 cc/25

kg BW. Ventral Surface: In the central region of the oral cone the
tegument was severely swollen and the spines were barely visible
(arrows).

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has been reported for salicylanilides, rafoxanide, and

closantel, with cross resistance to nitroxynil. Another
report on treatment with closantel and nitroxynil was
published in SCOPS[41].
It was postulated that the administration of these
flukicides to animals used for human consumption
can lead to transfer of drug residues to human
food[12,21,42]. In 2013, all drug products containing
the active ingredients of clorsulon, closantel, nitroxynil,
rafoxanide, and TCBZ were prohibited for use
in lactating animals producing milk for human
consumption. The reason presented was that residues
in undetectable concentrations in milk could be
concentrated during manufacturing, and become

detectable in milk products [12]. The researchers

recommended treatment of dairy cows during the dry
period, when no milk is being produced for human
consumption, and advised that an adequate withdrawal
period is allowed for the elimination of drug residues
before resumption of milking.
In vitro studies have shown great drug disruption
of worms following treatment with clorsulon and
nitroxynil [33,43]. Besides ingestion through the gut,
the tegument presents an absorptive surface for the
uptake of drugs by the fluke. The adult fluke is bathed
in bile when in the bile ducts of the host, which are
its preferential dwelling site, and becomes exposed to
drugs excreted in the flow of bile. Thus, because the

Figure 3

Figure 4

SEM of F. gigantica after treatment in vivo with Nitroxynil 1 cc/25

kg BW. Ventral Surface: In the anterior mid-body region, directly
behind the ventral sucker, no spines were visible as they had become
completely submerged by the extremely swollen tegument.

SEM of F. gigantica after treatment in vivo with Nitroxynil 1 cc/25 kg

BW. Ventral Surface: In the mid-body region, the spines appeared
almost submerged by the swollen tegument surrounding them.

Figure 5

SEM of F. gigantica after treatment in vivo with Nitroxynil 1 cc/25

kg BW. Dorsal Surface: The spines appeared to be flaking off, with
multiple pores and furrowing of the tegument (arrows).

Figure 6

Identified SEM of F. hepatica after treatment in vivo with Nitroxynil

1 cc/25 kg BW. Ventral surface. Tegumental swelling of the apical
cone, showed regional variations in severity surrounding the oral and
ventral suckers (arrows).

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Effect of nitroxynil on adult Fasciola Omran and Ahmad

111

Figure 7

Figure 8

SEM of F. hepatica after treatment in vivo with Nitroxynil 1 cc/25 kg

BW. Ventral surface. Identified area between the oral and ventral
sucker showed displaced spines (arrow).

SEM of F. hepatica after treatment in vivo with Nitroxynil 1 cc/25 kg

BW. Ventral surface. The mid-body region laterally showed displaced
spines directly behind the ventral sucker, no spines were visible as
they had become completely submerged by the extremely swollen
tegument.

Figure 9
Figure 10

SEM of F. hepatica after treatment in vivo with Nitroxynil 1 cc/25 kg

BW. Ventral surface. The central mid-body region showed severe
swelling and furrowing of the tegument (arrow).

tegument is immediately exposed to anthelminthics,

it is a crucial drug target. The drug-induced damage of
the tegument facilitates drug penetration to deeperlying tissues, thus leading to serious effects on the
flukes. It was deduced by McKinstry et al. [33] that,
in the in vivo situation, the surfactant action of bile
and the immune response aggravates the damage,
leading to the discoloration of the flukes observed
after 72 h treatment. Nitroxynil is excreted mostly
unchanged through the liver giving rise to high
concentrations in the bile ducts. Recent research
showed that a metabolite of nitroxynil produced in
the liver parenchyma added to its flukicidal activity
and explained its efficacy against late immature flukes
that migrate through the liver tissues [34].

SEM of F. hepatica after treatment in vivo with Nitroxynil 1 cc/25 kg

BW. Dorsal surface. The spines appeared to be flaking off with multiple
pores and furrowing of the tegument (arrow).

In the present study, the flukes recovered from the

bile ducts of nitroxynil-treated cows 15 days after
treatment were not dead and moved sluggishly. They
appeared pale with no evidence of gut contents. As
described, healthy flukes are grayish-brown in color,
with a flattened spiny tegument, through which
the dark outline of blood-filled ceca is evident [44].
The pallor and empty guts of flukes, observed in our
study confirms that penetration and autophagia of
substances such as bile and drugs takes place [32].
As regards other flukicides, it was reported that
praziquantel produces flaccid paralysis of the fluke,
starting with initial increase followed by decrease
in muscle tone [45]. The researchers added that

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oxyclozanide, rafoxanide, niclofolan, bithionol, and

hexachlorophene also induce rapid spastic paralysis of
the fluke. Nitroxynil produces a similar effect, which
explains the sluggish movement recorded in our study.
By discontinuing oxidative phosphorylation in the cell
mitochondria, it disrupts the production of ATP, thus
causing a long-term suppression of movement [23,24].
In another report, the flukes after treatment in vivo
with nitroxynil showed little or no sign of movement
and appeared yellow in color, with no evidence of gut
contents. Swelling of the mid-body was visible to the
naked eye [36].
The present study has confirmed that nitroxynil causes
swelling and severe disruption of the tegumental
surface of Fasciola, as well as swelling of the basal
in-folds in the tegumental syncytium, edematous
flooding of the parenchyma, and mitochondrial
deformation in the syncytium and tegumental
cells[46,47]. In our study, no spines were visible in the
anterior mid-body region, directly behind the ventral
sucker, as they had become completely submerged
by the externally swollen tegument. The mid-body
region also displayed widespread swelling and
extensive furrowing of the tegument, and the spines
were almost submerged by the swollen tegument
surrounding them. From previous studies, disruption
to the spines seems to be a particular feature of
nitroxynil action [33,44,45]. When due to the action
of the drug, the thin tegument covering the spines
splits and sloughs off and the spines are lost, the holes
thus formed in the syncytium allow more access of
the drug to internal tissues. This becomes especially
promoted by the surfactant action of bile in vivo [33].
The cause for disruption of spine structure observed
in our study and reported with other fasciolicides is
unknown [4850].
In the present study, the dorsal surface disruption
was as severe as that seen on the ventral surface.
The spines appeared to be flaking off, and multiple
pores and furrowing of the tegument were detected.
This outcome of in vivo treatment with nitroxynil
was previously defined as regional differences in
tegumental disruption that is more severe on the
dorsal than on the ventral surface, and on the anterior
than on the posterior region of the fluke [33]. In their
investigation, the researchers also described extensive
swelling and blebbing of the tegument on both
surfaces, and at high magnification microvillus-like
projections were evident with some tegumental loss
in the oral region of the fluke. In our point of view,
greater affection of the dorsal surface resulted from
higher exposure to the drug as the flukes are folded
ventrally when in the bileducts.

McKinstry et al. [36] stated that, after 24 h of in vivo

treatment with 40 mg/kg of nitroxynil, the recovered
flukes were moving normally but appeared pale,
with no evidence of gut contents. After 48 h, the
flukes were less active, and surface swelling could be
observed. After 72 h, the flukes showed little or no
sign of movement; all appeared yellow with empty
gut contents. Swelling of the mid-body was visible to
the naked eye. In comparison, another report found
that after the full course of in vivo treatment with
TCBZ the recovered flukes remained active for 48 h
displaying limited morphological disruption, but they
were all dead after 72 h. The researchers described some
blebbing and sloughing of the tegument around the
oral sucker, when an extra layer had apparently been
deposited on the fluke surface, giving it a flattened
appearance [51]. A relatively recent investigation
indicated that each of TCBZ-susceptible and
TCBZ-resistant isolates were affected by nitroxynil
treatment in vitro and in vivo, showing ultrastructural
changes similar to those previously seen in the
Cullompton TCBZ-susceptible isolate [36]. In the
present study, 15 days after treatment the flukes were
still alive, but suffered severe swelling of the tegument,
accompanied by isolated areas of flattening along the
lateral margins of the flukes and in the tail region.
Our SEM examination of nitroxynil-treated flukes
showed limited areas of tegument sloughing in the
tail region. In more seriously affected specimens, the
syncytium appeared stripped away to reveal the basal
lamina. Some deeper lesions were also observed as the
loss of tegument facilitated penetration of the drug
to the exposed deeper tissues of the flukes. There was
disruption of the subtegumental tissues with spacing
or flooding between the cells and breakdown of the
cell bodies, to the extent that often they were difficult
to identify. Similar findings were described [32] in
TCBZ-treated flukes. The flooding feature was also
recorded in other drug studies on F. hepatica using
sulfoxide metabolite of the compound alpha [50] and
metabolites of TCBZ as sulfoxide (TCBZSO) and
sulfone (TCBZSO2) [52]. A very recent publication
in 2015 [53] revealed that, in sheep with a high fluke
burden, TCBZ was ineffective in treating chronic
fasciolosis indicating TCBZ resistance. At the same
time nitroxynil and closantel proved to be wholly
active against TCBZ-resistant flukes in chronically
infected sheep with high chronic fluke burdens.
In conclusion, our study has confirmed previous SEM
studies with nitroxynil in that the drug effectively
disrupts the tegument of Fasciola. In addition, it causes
disruption to the gut following in vivo treatment, an
observation that is consistent with the oral uptake of
the drug as well. Giving similar therapeutic results
with little resistance, nitroxynil therapy of fascioliasis

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Effect of nitroxynil on adult Fasciola Omran and Ahmad

in rural animals may be considered a safe replacement

for TCBZ treatment.

113

20 Borgsteede FHM, Moll L, Vellema P, Gaasenbeek CPH. Lack of reversion

in triclabendazole resistant F. hepatica. Vet Rec 2005; 156:350351.

Acknowledgements

21 Whelan M, Kinsella B, Furey A, Moloney M, Cantwell H, Lehotay SJ,

Danaher M. Determination of anthelmintic drug residues in milk using ultra
high performance liquid chromatography-tandem mass spectrometry with
rapid polarity switching. J Chromatogr A 2010; 1217:46124622.

Author contribution: EK Omran and NS Ahmad proposed

the research idea, conducted the work, and shared in writing
and revision of the manuscript.

22 Ghoneim MM, El-Ries M, Hassanein AM, Abd-Elaziz AM. Voltammetric

assay of the anthelminthic veterinary drug nitroxynil in bulk form and
formulation at a mercury electrode. J Pharm Biomed Anal 2006;
41:12681273.
23 Boray JC, Happich A. Standardized hemotherapeutical tests for immature
and mature F. hepatica infections in sheep. Aust Vet J 1968; 44:7278.

Nil.

24 Rapic D, Dzakula N, Sakar D, Richards RJ. Comparative efficacy of

triclabendazole, nitroxynil and rafoxanide against immature and mature F.
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Conflicts of interest

25 Alvinerie M, Floch R, Galtier P. Plasma protein binding of nitroxynil in

several species. J Vet Pharmacol Ther 1991; 14:170173.

Financial support and sponsorship

There are no conflicts of interest.

26 Martin RJ. Modes of action of anthelmintic drugs. Vet J 1997; 154:1134.

27 Coles GC, Stafford KA. The activity of oxyclozanide, nitroxynil, clorsulon
and albendazole against adult triclabendazole resistant F. hepatica. Vet
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