Veterinary Parasitology 197 (2013) 522526

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

The use of nitazoxanide against the pathogens

Ichthyophthirius multiliis and Aeromonas hydrophila in silver
catsh (Rhamdia quelen)
Fernando J. Sutilia, Leticia T. Gresslerb, Agueda C. Vargasb,
Carla C. Zeppenfeld a, Bernardo Baldisserotto a, Mauro A. Cunhaa,
a
Laboratrio de Fisiologia de Peixes-(LAFIPE), Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, Santa
Maria, RS BR-97105-900, Brazil
b
Laboratrio de Bacteriologia-(LABAC), Departamento de Medicina Veterinria Preventiva, Universidade Federal de Santa Maria, Santa
Maria, RS BR-97105-900, Brazil

a rti c l e in f o abstrac t

Article history: The antiparasitic and antibacterial activities of nitazoxanide (NTZ) against Ichthyophthirius
Received 5 February 2013 multiliis (Ich) and Aeromonas hydrophila, respectively, were evaluated in silver catsh juve-
Received in revised form 6 June 2013
niles (Rhamdia quelen). In the rst experiment, sh naturally infected by Ich were treated
Accepted 11 June 2013
with different concentrations of NTZ. Fish mortality and the number of trophonts/juveniles
were evaluated. In the second experiment, the antimicrobial potential in vitro (MIC test)
Keywords:
and preventive potential in vivo against A. hydrophila was evaluated. The treatment with
Ich
Antiprotozoal 1.5 mg L1 NTZ showed the best result against Ich, with a signicant reduction of trophonts
Ichthyophthiriasis and sh mortality of 10%. NTZ did not show in vitro activity against A. hydrophila at concen-
Antimicrobial trations tested, but showed a probable preventive activity in vivo. However, these results
are preliminary and more studies should be conducted.
2013 Elsevier B.V. All rights reserved.

1. Introduction economic losses to the aquaculture industry. The life cycle

In aquaculture systems sh are sometimes concomi-
tantly infected by multiple disease agents and co-infections
by parasite and bacteria contribute to the severity of
some diseases. Ichthyophthirius multiliis and Aeromonas
hydrophila are two common pathogens of cultured sh
(Xu et al., 2012a). The protozoan I. multiliis is a para-
sitic ciliate (commonly referred to as Ich) that infects a
range of freshwater sh species and causes the disease
known as ichthyophthiriasis. It is one of the most important
pathogenic parasites of cultured sh, causing signicant
Corresponding author. Tel.: +55 553320 9382; fax: +55 55 3220 8241.
E-mail addresses: cunha.mauroalves@gmail.com,
mauroalves @hotmail.com (M.A. Cunha).
of Ich consists of three stages: an infective theront, a par-
asitic trophont, and a reproductive tomont (Matthews,
2005). Outbreaks of bacteria of the genus Aeromonas in sh
are associated with changes in environmental conditions
and/or stress factors like parasitic infections (Barcellos
et al., 2008). Infections caused by Ich enhance potential of
invasion by A. hydrophila in sh causing outbreaks in sh
farms with high mortality (Xu et al., 2012a).
Silver catsh (Rhamdia quelen) is a fast-growing species
native to South Brazil which is well-adapted to cultiva-
tion. The use of salt, copper sulphate, and temperature rise
are reported as alternatives to Ich control in silver catsh,
but these treatments are limited by factors such as toxic-
ity, as in the case of copper sulfate or are uneconomical in
most sh culture conditions (Carneiro et al., 2005). Various
antimicrobial drugs have been used against A. hydrophila,

0304-4017/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetpar.2013.06.012
 F.J. Sutili et al. / Veterinary Parasitology 197 (2013) 522526
but the undue use of these drugs in silver catsh culture
resulted in the emergence of resistant strains (Barcellos
et al., 2008).
Nitazoxanide [2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]
phenyl] acetate (NTZ) has broad-spectrum antiparasitic
activity, acting in a wide spectrum of protozoa parasites in
humans, such as amoebas, agellates, sporozoans and cili-
ates (Abaza et al., 1998), as well as bacteria (Dubreuil et al.,
1996; de Carvalho et al., 2011). Therefore, based on the
needs of a therapy against ichthyophthiriasis and infections
by A. hydrophila, the antiparasitic and antibacterial poten-
tial of NTZ was analyzed for the rst time in sh infected
by these pathogens.
2. Materials and methods
2.1. Animals and water quality
Juvenile silver catsh (9.86 0.40 g; 10.61 0.18 cm)
were purchased from a local sh culture and transported to
the Laboratrio de Fisiologia de Peixes at the Universi-
dade Federal de Santa Maria, where they were acclimated
for seven days in continuously aerated 250 L fresh water
tanks with controlled water parameters (20.8 0.04 C, pH:
7.67 0.04, dissolved oxygen levels: 7.34 0.27 mg L1).
Dissolved oxygen and temperature were measured with an
YSI oxygen meter (Model Y5512). The pH was determined
with DMPH-2 pH meter (Digimed). Total ammonia levels
were determined according to Verdouw et al. (1978) and
un-ionized ammonia (NH3) levels were calculated accord-
ing to Colt (2002).
2.2. Toxicity test
To evaluate drug toxicity 50 uninfected silver catsh
juveniles were exposed to different NTZ concentrations
(Annita, Farmoquimica), (0.0-control, 1.0, 1.5, 2.0, or
3.0 mg L 1, in duplicate, n = 5 each) using 10 L aquaria. A
single drug application was made in water and after 48 h
30% of the water was replaced by the same volume with-
out the drug. Fish were fed to satiation once a day with
commercial concentrate for juveniles with 42% extruded
crude protein (Alisul/Supra Brazil). Uneaten food as well
as other residues and feces were removed out 30 min after
feeding. Dead sh were also removed daily.
2.3. Experiment 1- Ichthyophthirius multiliis
A semi-static system was used, and 150 sh (in tripli-
cate, n = 10 each) naturally infected with I. multiliis were
transferred to 10 L aquaria. Fish were submitted to the same
protocol and NTZ concentrations mentioned in Section 2.2.
A single drug application was made in water according
mentioned in Section 2.2.
The infective trophonts were counted in 50% of
sh, at 0 and 48 h and 100% of sh at 96 h. Fish
were then removed from the treatments and remained
under observation up to 144 h. The cumulative mortal-
ity rate was calculated from the number of dead sh
removed from each aquarium throughout the experi-
mental period. Fish were anaesthetized with 40 L L1
523
eugenol (Cunha et al., 2010) and the number of trophonts
was determined with the assistance of a stereomicro-
scope (total magnication 10) according to Bush et al.
(1997).
During the experiment, sh from the control group
presented ulcerative lesions on the skin and a possible con-
comitant bacterial infection was evaluated. Fish from other
groups did not show any lesion on the skin, but were also
sent for analysis. The bacterial isolation was made from
lesions and kidneys of two sh from each treatment at 96 h.
Morphological and biochemical characteristics were ana-
lyzed according to Quinn (1994) for bacterial identication
and characterization.
2.4. Experiment 2Aeromonas hydrophila
A second experiment was conducted to evaluate the
in vitro activity of NTZ against A. hydrophila. Test for
the Minimum Inhibitory Concentration (MIC) of NTZ was
conducted in anaerobic and aerobic conditions, using ster-
ile 96-well polystyrene microtiterplates by microdilution
(CLSI, 2008). Aeromonas hydrophila ATCC 7966 and a strain
isolated from the sh were used in the test. The concen-
trations tested were 0.75, 1.5, 3.0, 6.0 and 12.0 g mL1 (in
triplicate).
An in vivo test was conducted to investigate the
potential of NTZ in preventing bacterial infection by A.
hydrophila in sh parasited by Ich. Thirty six sh nat-
urally infected with I. multiliis were transferred to 6 L
aquaria divided into two treatments: 0.0-control and
1.0 mg L1 of NTZ (concentration without effect against
Ich in the previous experiment), in triplicate (n = 6 each)
with 52.2 10.6 and 60.5 13.7 trophonts/sh, respec-
tively. A single drug application was made in the water
and after 48 h 30% of the water was replaced by the
same volume without the drug. At 96 h the sh were
removed from the treatments and remained under obser-
vation for four additional days. After drug application (0 h)
both groups were challenged with an addition of a bacte-
rial solution (2 mL) containing 2.0 109 CFU mL1 in each
aquarium (A. hydrophila, ATCC 7966). The sh were fed
to satiation once a day. Uneaten food as well as other
residues and feces were removed out 30 min after feed-
ing.
The cumulative mortality rate was calculated from
the number of dead sh removed from each aquarium
throughout the experimental period. The development and
counting of lesions was observed at 48 and 96 h. The count-
ing of sh with lesions was performed only in the treatment
period (096 h) because sh mortality occurred through
the period of observation (96192 h). The lesions occurred
in the dorsal, pelvic and pectoral ns and maxillary bar-
bells. Partial or total loss of maxillary barbells, frayed and
ulcerated ns and tissue loss between n rays with hemor-
rhage were observed and considered. Each of the following
situations was counted as a lesion: (i) lesion on the dorsal
n; (ii) lesion in one or both pectoral ns; (iii) lesion in one
or both pelvic ns; (iv) lesion in one or both maxillary bar-
bells. Fish with at least one of these lesions was considered
for counting.
524 F.J. Sutili et al. / Veterinary Parasitology 197 (2013) 522526

The methodology of these experiments was approved

by the Ethical and Animal Welfare Committee of the Uni-
versidade Federal de Santa Maria (Process n 046/2010).

2.5. Statistical analysis

The homogeneity of variances among groups was tested

with the Levene test. Number of trophonts in all treat-
ment groups was compared using one-way ANOVA and
Tukey test and number of sh with lesions was compared
by MannWhitney U test (Statistica 7.0 Software). Fish
survival was compared by KaplanMeier survival analy-
sis with Logrank Test (SPSS 18 Software). The minimum
signicance level was set at P < 0.05.

3. Results

3.1. Water quality

The parameters of water quality were: temperature

20.0 0.04 C, dissolved oxygen levels 7.69 0.12 mg L1,
pH 7.34 0.05, maximum total ammonia 2.4 0.9 mg L1
and maximum non-ionized ammonia 0.02 0.007 mg L1.

3.2. Toxicity test

Fish mortality (20%) was observed only at the highest

concentration (3.0 mg L1).

3.3. Experiment 1
The survival in the control group was 40% after 144 h
of experiment. In groups treated with 1.0 and 1.5 mg L1
survival was 97% and 90% respectively, but in the groups
treated with 2.0 and 3.0 mg L1 of NTZ, despite a reduc-
tion in the number of trophonts, survival was 76% and
50%, respectively (Fig. 1A). Fish treated with 1.0, 1.5, 2.0
or 3.0 mg L1 NTZ showed a signicant reduction in the
amount of trophonts/sh at 48 h compared to 0 h and to
the control group in the same period. After 96 hall surviving
sh (except those treated with 1.0 mg L1 NTZ) showed a
signicant reduction in the number of trophonts compared
to 48 h. Fish exposed to 1.5, 2.0 or 3.0 mg L1 NTZ presented
reduction of approximately 98% in the number of trophonts
compared to 0 h. However, those treated with 1.0 mg L1
NTZ showed an increase in the number of trophonts, dif-
fering signicantly from the other groups at 96 h (Fig. 1B).
Control dead sh showed typical signs of bacterial infec-
tion, mainly small ulcerative lesions in the ns. In these sh,
infection by bacteria of genus Aeromonas sp. was found. Fish
exposed to 1.0 mg L1 NTZ presented the highest number
of trophonts, but did not show any sign of bacterial infec-
tion. Aeromonas sp. was not isolated from any group besides
control.
3.4. Experiment 2
The MIC test showed that NTZ at concentrations lower
than 12 g mL1 has no inhibitory activity against the
strains of A. hydrophila tested in anaerobic and aerobic
conditions. In the in vivo experiment, survival was higher
Fig. 1. (A) Survival of silver catsh infected with I. multiliis and treated
with different nitazoxanide concentrations. (*) indicates signicant differ-
ence relative to control. KaplanMeier survival analysis with Logrank test
(P < 0.05). (B) Nitazoxanide effect on Ich trophonts as a function of time.
(*) indicates signicant difference relative to 0 h. (#) indicates signicant
difference relative to the other treatments at the same period. One-way
ANOVA and Tukey test (P < 0.05).
(100%) in sh treated with 1.0 mg L 1 NTZ than in control
(Fig. 2A). Fish showed the rst lesions in less than 48 h. At
48 h the percentage of sh with lesions was signicantly
lower in the treatment with NTZ, but at 96 h there was
no signicant difference between groups (Fig. 2B). At the
end of the experiment the lesions in the sh ns exposed
to 1.0 mg L 1 of NTZ had regressed and only lesions in
the barbells remained. The number of trophonts/sh was
not changed by NTZ treatment throughout this experi-
ment. Aeromonas sp. was re-isolated from the lesions and
kidneys of dead control sh and identied as detailed in
experiment 1.
4. Discussion
Several drugs such as formaldehyde, sodium chloride
(salt), copper sulphate and potassium permanganate are
currently administered in the form of baths against Ich
infections (see review of Picn-Camacho et al., 2011).
In many countries it is no longer allowed to treat sh
for human consumption with malachite green, due to its
carcinogenic potential (Wohllebe et al., 2012). Formalde-
hyde was reclassied by the World Health Organization
 F.J. Sutili et al. / Veterinary Parasitology 197 (2013) 522526
Fig. 2. Nitazoxanide effect on silver catsh infected with A. hydrophila and
I. multiliis. (A) Survival. KaplanMeier survival analysis with Logrank test
(P < 0.05). (B) Number of sh with lesions. MannWhitney U test (P < 0.05).
(*) indicates statistically signicant difference from control at the same
period.
International Agency for Cancer Research as carcino-
genic to humans (WHO IARC, 2006). Nitazoxanide is
approved for human use by the US Food and Drug Admin-
istration (FDA) for the treatment of diarrhea caused by
Cryptosporidium species and Giardia intestinalis and has
been studied worldwide (Fox and Saravolatz, 2005), but
this drug is not mentioned and permitted by European
Medicines Agency (EMA) (see European Medicines Agency
website).
Drug employed for the reduction of Ich can be divided
principally into two groups: those that target and have
an effect on the parasitic trophont stage within the host
epithelium, and those targeting the free-living external
stages of the parasite (Matthews, 2005). According to
Picn-Camacho et al. (2012) the majority of treatments
fall into the latter category. Nitazoxanide is absorbed by
the organism and metabolized to form the active metabo-
lite, tizoxanide (Gilles and Hoffman, 2002). Since Ich xes
between the dermis and epidermis of sh and feeds on tis-
sue uids and fragments of cells (Pavanelli et al., 2002),
525
probably NTZ acts at the parasitic stage of the Ich. The treat-
ment with the lowest concentration (1 mg L1) reduced the
number of trophonts in 48 h, but at 96 h sh in this treat-
ment increased their number of white spots. Apparently,
the dilution of this concentration after 48 h impaired the Ich
treatment in silver catsh. The reduction of the number of
white spots was gradual at the highest concentrations spite
of the dilution at 48 h.
Metronidazole, an antiparasitic with a similar mecha-
nism of action to NTZ against protozoa, was very successful
at reducing the number of trophonts on infected sh when
added to diets (Toks en and Nemli, 2010). This com-
pound is effective in ornamental sh, but is currently
listed as possibly carcinogenic to humans by the World
Health Organization (Picn-Camacho et al., 2011). The
present experiment showed that infected sh survival was
inversely proportional to the concentrations of NTZ. In the
toxicity test healthy sh mortality occurred only at the
highest concentration. The NTZ was effective against the
parasite, but has a narrow safety margin in silver catsh.
The drug was toxic to sh weakened by Ich.
The dead control silver catsh (experiment 1) pre-
sented infection by Aeromonas sp. Bacteria of this genus
are typically opportunistic and manifest themselves
in hosts attacked and weakened by other etiological
agents (Pavanelli et al., 2002). Ich is a potential vector
for pathogenic bacteria of sh, such as Flavobacterium
columnare (Sun et al., 2009) and Ewardsiella ictaluri (Xu
et al., 2012b). Silver catsh parasitized treated with the
lowest NTZ concentration did not present natural infection
by Aeromonas sp. The results suggest that control sh mor-
tality is largely due to the co-infection between Aeromonas
and Ich. In the group treated with 1.0 mg L 1 survival was
very high after 96 h, but there was no effect on ciliates
viability.
Nitazoxanide is effective against several gram-positive
and gram-negative bacteria, specically Bacteroides and
Clostridium species, Helicobacter pylori, Mycobacterium
tuberculosis and bacteria of genus Staphylococcus (Dubreuil
et al., 1996; Megraud et al., 1998; Tchouaf-Nana et al.,
2010; de Carvalho et al., 2011). In this study NTZ did
not show inhibitory activity in vitro against A. hydrophila.
In the prophylactic trial, silver catsh exposed to NTZ
showed a signicant lower number of lesions at 48 h and
100% survival after bacterial challenge, indicating a prob-
able preventive effect. In sub-inhibitory concentrations
(MIC) NTZ has inhibitory effect on biolm formation in
bacteria of genus Staphylococcus and Escherichia coli, and
hemagglutination inhibition in E. coli (Shamir et al., 2010;
Tchouaf-Nana et al., 2010). Antimicrobial agents that
inhibit virulence factors can be good candidates to prevent
infections.
With a broad spectrum of action against protozoa and
bacteria, a possible activity of NTZ against pathogenic
bacteria of sh such as those from the genus Aeromonas
and other important bacteria in sh farming should be con-
sidered and further mechanisms of action of NTZ must be
examined.
In conclusion, results showed that a single application
of NTZ was effective against the parasite Ich. The best
result was obtained with the treatment of 1.5 mg L1 that
526 F.J. Sutili et al. / Veterinary Parasitology 197 (2013) 522526
provided a signicant reduction of trophonts and survival
of 90% of sh. Against A. hydrophila NTZ did not show
activity in vitro in concentrations lower than 12 g mL1,
but showed an apparent preventive potential in vivo.
These results are preliminary and more studies should be
conducted to evaluate a greater number of applications
or longer exposure period, enabling a greater efciency
and/or reduction of the effective concentration.
Conict of interest statement
None of the authors has any nancial or personal rela-
tionships that could inappropriately inuence or bias the
content of the paper.
Acknowledgments
The authors acknowledge support from CNPq (Conselho
Nacional de Desenvolvimento Cientco e Tecnolgico),
process 470964/2009-0. B. Baldisserotto received a CNPq
research fellowship, and F. J. Sutili and M.A. Cunha received
a CAPES (Coordenac o de Aperfeic oamento de Pessoal de
Nvel Superior) Msc and Postdoctoral fellowships, respec-
tively.
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