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Contents lists available at ScienceDirect

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

Efcacy of garlic based treatments against monogenean

parasites infecting the guppy (Poecilia reticulata (Peters))
S. Fridman , T. Sinai, D. Zilberg
French Associates Institute for Agriculture and Biotechnology of Drylands, Ben Gurion University of the Negev, Sde Boqer Campus, Beer
Sheva 84990, Israel

a r t i c l e i n f o a b s t r a c t

Article history: Monogenean infections of commercially farmed shes are responsible for signicant
Received 23 September 2013 economic losses. Garlic (Allium sativum) is a well-known spice which also possesses
Received in revised form 30 January 2014 anti-microbial and anti-parasitical properties. The current work aimed to test the efcacy
Accepted 4 February 2014
of garlic-based treatments against infection with monogenean sp. in the guppy (Poecilia
reticulata). Clipped sections of tail ns of guppies heavily infected with Gyrodactylus
Keywords:
turnbulli were exposed to aqueous garlic extract (7.5 to 30 mL L1 ) and visually observed
Natural treatments under a dissecting microscope. Results revealed that exposure to garlic caused detachment
Garlic of parasite and cessation of movement indicating death. A positive correlation was seen
Ornamental shes between garlic concentration and time to detachment and death of parasites, which, at
Monogenean the highest concentration of 30 mL L1 , occurred at 4.1 and 8.6 min, respectively. Bathing
in aqueous garlic extract (7.5 and 12.5 mL L1 ) was tested in guppies infected with G.
turnbulli. Prior acute toxicity tests revealed the maximum tolerance levels of guppies to
garlic extract to be 12.5 mL L1 for 1 h. Bathing of infected sh in garlic extract (7.5 and
12.5 mL L1 ) signicantly (p < 0.05) reduced infection prevalence and intensity as compared
to the control. Oral treatments using dry garlic powder-supplemented diet were tested on
guppies infected with G. turnbulli and Dactylogyrus sp. Fish were fed with food containing
10% and 20% dry garlic powder for 14 days. Groups fed with garlic supplemented diets
showed signicantly reduced (p < 0.05) mean prevalence and mean intensity of parasites as
compared to the control. Dietary application of garlic did not appear to affect palatability.
Fresh crushed garlic was added at a level of 1 g L1 and applied as an indenite bath for
14 days. This treatment was seen to signicantly reduce (p < 0.05) parasite prevalence and
mean intensity as compared to the control. Histopathology revealed elevated muscular
dystrophy in the 20% garlic-fed group, as compared to control. These ndings demonstrate
the potential of garlic as a natural alternative to currently used chemical treatments for
monogenean sp. infection in the guppy.
2014 Published by Elsevier B.V.

1. Introduction

Since 1985, the value of international trade in exports

Corresponding author. Tel.: +44 972 8 6596838/+44 972 5 86278697;
fax: +972 8 6596742.
E-mail address: sfridman@post.bgu.ac.il (S. Fridman).
of ornamental sh species has increased at an average
growth rate of approximately 14% per year (FAO, 2010).
Such a rapidly expanding sector has the potential to
contribute to the economic growth of the ornamental
aquaculture industry as a whole, however, occurrences of

http://dx.doi.org/10.1016/j.vetpar.2014.02.002
0304-4017/ 2014 Published by Elsevier B.V.

Please cite this article in press as: Fridman, S., et al., Efcacy of garlic based treatments against monogenean parasites
infecting the guppy (Poecilia reticulata (Peters)). Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.002
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large-scale, parasite-related sh diseases causing signif-
icant economic losses threaten long-term sustainability.
The guppy (Poecilia reticulata), a tropical, freshwater sh
originating from the Caribbean and Central and South
America, is an important cultured ornamental species. Its
popularity with aquarists is due to its ease of maintenance
and its attractive and varied colouration.
Guppies are natural hosts for a range of monogenean
parasites that inhabit the skin, ns, gills, pseudobranchs
and buccal cavities (Bakke et al., 2002). Gyrodactylus sp.
are monogenean trematodes with a direct life-cycle; they
reproduce directly onto the hosts skin with no interme-
diate stage and every individual is born with a pregnant
embryo in its uterus, therefore population growth can be
high (Lyles, 1990). Transmission amongst host individuals
is either by direct contact between sh or via the water col-
umn or substrate (Scott and Anderson, 1984; Solenge et al.,
1998). Dactylogyrus sp. are oviparous monogeneans usually
found on gills of host shes. Possessing a direct life-cycle,
oviparous adults produce eggs directly into the water col-
umn. After hatching, water currents aid the free-swimming
ciliated larva to attach to the gills of a sh host. Heavy infes-
tations of the gills may cause severe hyperplasia of the gill
lament epithelium, interfering with respiratory function,
and may be a direct cause of death (Paperna, 1991).
Although in the natural environment monogeneans
cause few apparent problems, their short, direct, one-host
life cycle combined with the unnaturally high concen-
tration of host shes in an intensive aquaculture system
allows infestations to quickly reach epizootic levels. Dam-
age is caused mainly by the mechanical action of their
attachment apparatus. Studies elucidating host responses
against gyrodactylids suggest the probable involvement of
both innate and acquired immune components (Buchmann
et al., 2004; Cable and van Oosterhout, 2007). Innate
defense mechanisms, such as the proliferation of mucous
cells, play a pivotal role in host responses against gyro-
dactylids, as complement and other immune components
reach the sh surface via mucus (Buchmann et al., 2004).
Severe infestations, causing a combination of inhibition of
respiratory gas exchange and osmotic regulation and sec-
ondary infection by bacteria or fungi, usually result in death
(Paperna, 1991).
Monogeneans are notoriously difcult to control and
various chemical treatments have been used against these
parasites, primarily by immersive or bath treatment appli-
cation. Most theurapeutants, however, pose associated
problems such as low efcacy, host toxicity and environ-
mental and human health concerns (see review by Schelkle
et al. (2009)). In addition, antihelminthic-resistant strains
of parasites can develop (Goven and Amend, 1982; Schmahl
et al., 1989). As witness to the current trend to move away
from chemical treatments in aquaculture, there has been
an associated increase in interest and research into the uti-
lisation and efcacy of traditional plant-based medicines
to control diseases in shes. Garlic (Allium sativum) is an
edible plant which historically has been used as a medic-
inal remedy to treat a wide range of conditions (Ankri
and Mirelman, 1999). Pena et al. (1988) rst reported
anti-helmintic properties of crushed garlic against Capil-
laria sp. in the common carp (Cyprinus carpio), and more
Please cite this article in press as: Fridman, S., et al., Efcacy of garlic based treatments against monogenean parasites
infecting the guppy (Poecilia reticulata (Peters)). Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.002
recently, the inhibitory effects of garlic-derived products
for sh parasite infestations has been reported against the
freshwater protozoan parasite Ichthyophthirius multiliis
(Buchmann et al., 2003), Spironucleus vortens (Millet et al.,
2010; Williams et al., 2012), gyrodactylosis and trichodi-
nosis in the Nile tilapia (Oreochromis niloticus) (Abd El-Galil
and Aboelhadid, 2012), Gyrodactylus turnbulli in the guppy
(P. reticulata) Schelkle et al. (2013) and the monogenean
Neobenedenia sp. in farmed barramundi (Lates calcarifer)
(Militz et al., 2013a, b). In more recent work, the value of
dietary dosages of garlic in promoting immune response,
disease resistance and survival in various shes following
bacterial challenge has been documented e.g. Aeromonas
hydrophila in the Nile tilapia (O. niloticus) (Aly et al., 2010),
A. hydrophila in the rainbow trout (Oncorhynchus mykiss)
(Nya and Austin, 2009), A. hydrophila in the Rohu (Labeo
rohita) (Sahu et al., 2007) and Vibrio harveyi in the barra-
mundi (L. calcarifer) (Talpur and Ikhwanuddin, 2012).
This study examines the anti-parasitic effect of gar-
lic against Monogenean parasites infecting guppies. The
efcacy of aqueous garlic extract was tested in vitro and
in vivo. Additionally, treatment of infected sh with garlic-
supplemented diets was investigated. Histopathological
changes associated with the garlic-supplemented diets
were characterised.
2. Materials and methods
2.1. Source of animals and propagation of parasites
Guppies, (0.40.06 g), were obtained from a commer-
cial sh farm. Upon arrival, a sample of sh with standard,
round caudal n tail design, were anaesthetised in 0.025%
clove oil (Kildea et al., 2004) and examined using a dis-
secting microscope (Zeiss Stemi 2000-C) for presence of
monogenean parasites. Once infection was conrmed, sh
were transferred to a 200 L tank equipped with aeration
and submerged biological lters. Water was prepared using
freshwater that had been de-chlorinated through the use
of 50 mg L1 of sodium thiosulphatepentahydrate (William
Blythe, Accrington, UK). Water temperature was main-
tained at 25 1 C. Fish were fed daily at 2% of their body
weight (Ocean Nutrition, Belgium). Uninfected sh were
added and sh stocking density was maintained at a high
level to encourage propagation of the parasite. Experi-
mental protocols were carried out in compliance with
the principles of biomedical research involving animals
obtained from the Ben Gurion University Committee for
the Ethical Care and Use of Animals, Ben Gurion University
of the Negev, Israel. Authorisation number; IL-79-10-2012.
2.2. Parasite identication
Gyrodactylid parasites were isolated from guppies
skin and ns and prepared for morphological analysis.
Individual parasites were placed on a glass slide and sub-
jected to proteolytic digestion using the method described
by Paladini et al. (2009) i.e. 3 L of digestion solution
(100 g mL1 proteinase K (Sigma Aldrich, US), 10 mM
EDTA (Sigma-Aldrich, US) and 5% SDS (Sigma-Aldrich,
US) added to each specimen with digestion continuously
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monitored under a dissecting microscope. Once complete,
the specimen was mounted using a cover slip and tissue
digestion was arrested with the addition of 1:1 saturated
ammonium picrate: 100% glycerin mix. The edges of the
cover slip were sealed using clear nail varnish. Parasites
were identied as G. turnbulli following the methods
of Harris et al. (1999). Dactylogyrus sp. was observed in
microscopic examination of fresh gill samples. Parasite was
identied to the genus level base on their morphological
characteristics, including the presence of four eyespots,
morphology of anchors and the absence of an embryo
(Mitchum, 1995).
2.3. Preparation of aqueous garlic extract
Fresh garlic was bought from a local supermarket and
a crude aqueous extract was prepared by grinding 10 g of
peeled garlic cloves in 50 mL distilled water in a domestic
blender. The stock extract was then ltered through What-
man no. 1 lter paper and stored at 4 C in a sealed bottle
and used within one month.
2.4. Experiment 1: In vitro parasite survival (7.5, 10,
12.5, 15, 20, 25, 30 mL L1 aqueous garlic extract)
The effect of garlic extract on in vitro parasite sur-
vival was tested. Guppies from the infection tanks were
selected, anaesthetised in 0.025% clove oil and examined as
described above for the presence of gyrodactylids. Heavily
parasitised sh were then euthanised by anesthesia fol-
lowed by pithing and areas of ns with a minimum of
three parasites were cut off using a scalpel. Each n clip
was transferred individually, using watchmakers forceps,
to a well of a 96-well plate (Corning Inc., USA) contain-
ing 1 mL ltered tank water. Parasites were observed for
movement using a dissecting microscope to ensure transfer
had not affected parasite survival. If any dead or moribund
parasites were observed, they were detached, if necessary,
from the n using a needle and removed using a Gilson
pipette. Garlic extract was then added to each well at 7.5,
10, 12.5, 15, 20, 25, 30 mL L1 and time was dened as zero.
Detached parasites are able to reattach to a host sh there-
fore both time to detachment and death were recorded.
Parasites were continuously observed and time to detach-
ment and death was noted. Non-motile parasites that did
not respond to a gentle water current created by moving a
needle through the water were considered dead. Fin clips
with parasites were immersed in 1 mL ltered tank water
without addition of garlic extract to serve as a control. A
minimum of 30 parasites were used for each treatment.
2.5. Experiment 2: Bath treatment of infected sh with
7.5 and 12.5 mL L1 aqueous garlic extract
2.5.1. Acute toxicity test
Preliminary trials were carried out to establish tolerance
levels of guppies to garlic extract; survival following expo-
sure for 1 h to 20 mL L1 garlic extract was 30% so it was
decided that 15 mL L1 should be the maximum concentra-
tion used. Ten guppies were added to 800 mL dechlorinated
freshwater with aeration in 1 L glass beakers and exposed
Please cite this article in press as: Fridman, S., et al., Efcacy of garlic based treatments against monogenean parasites
infecting the guppy (Poecilia reticulata (Peters)). Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.002
3
to varying concentrations of garlic extract (0, 7.5, 10, 12.5,
15 mL L1 ), each with three replicates. Mortality of sh was
recorded at 1, 2 and 6 h following continuous exposure.
2.5.2. Bath treatmentExperimental system
The efcacy of 1 h bath treatments using garlic extract
was tested. Guppies from the infection tanks were ran-
domly selected and placed in 1 L glass beakers containing
800 mL dechlorinated freshwater equipped with aeration.
A total of nine beakers were prepared, with three repli-
cates per treatment (n = 10 sh per replicate). To determine
infection rate at time 0, every sh from each beaker was
separately anaesthetised as before and examined under a
dissecting microscope in order to determine the number of
parasites from selected areas per sh; parasite counts were
made from both sides of caudal, dorsal and pectoral ns.
Fish were then revived and placed back in their 1 L glass
beakers and garlic extract was added at concentrations of 0,
7.5 or 12.5 mL L1 . Fish were exposed to the treatments for
1 h and then removed, anaesthetised and the parasite load
measured as before. Prevalence was calculated as the pro-
portion of infected hosts among all the hosts examined and
mean intensity was calculated as mean number of parasites
found in the infected hosts (according to Bush et al., 1997).
2.6. Experiment 3: Oral treatment with powdered garlic
2.6.1. Feed preparation
Fresh garlic, sourced as above, was peeled, dried at
60 C for 76 h and ground to a powder form in a domestic
grinder. The powder was carefully mixed with a com-
mercial, grained guppy food (Mem Ornamental, BernAqua,
Belgium) and vegetable oil (100 L/10 g food) was added
to ensure binding. Feed was stored in screw cap tubes at
4 C and used within one month.
2.6.2. Oral treatmentExperimental system
For Trial 1 guppies from the infection tanks
(0.54 0.07 g wet weight) were randomly selected
and distributed into four experimental groups with three
replicates per group (n = 40 sh per replicate). Each
replicate consisted of a 12 L freshwater aquaria, equipped
with aeration and submerged biological lters and water
temperature was maintained at 24.5 1 C. Treatments
included diets supplemented with 2% and 4% garlic powder
and a control diet without garlic was prepared with only
the addition of vegetable oil. For positive control, sh were
treated with Praziquantel, a conventionally recommended
treatment for monogenean infection, at 3 ppm at the start
(day 0) of the experiment, for 24 h, after which time the
water was replaced. These sh were fed the control diet.
For Trial 2 guppies from the infection tanks
(0.61 0.09 g wet weight) were randomly selected
and distributed into four experimental groups with three
replicates per group (n = 20 sh per replicate). Experi-
mental protocol was as for the rst trial, except that the
experimental diets contained 10% and 20% garlic powder.
Similarly a control diet without garlic was prepared with
only the addition of vegetable oil. An additional treatment
was fed the control diet and fresh chopped garlic at a rate
of 1 g L1 water (total 12 g/aquaria), enclosed in a small
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plastic container with holes (1 4 mm), was left in the

tank for the duration of the experiment as an indenite
bath.
For both trials, the sh were fed at 2% of body weight
once per day for a total of 14 days. Dead sh were removed
only after one day in order to allow the parasites to leave
the host and re-infect. At the start of each trial, a total of 10
sh were sampled from the infection tank and examined
for the presence of G. turnbulli. Fish were anaesthetised as
described before and the number of parasites on both sides
of the caudal tail was determined by direct examination
under a dissecting microscope. A skin scrape on one side
of the sh was performed and 3 gill arches were removed
and examined under a light microscope and the number of
parasites was counted. Gill examination revealed the pres-
ence of another monogenean (Dactylogyrus sp.), therefore
infection levels of this species were also recorded. At the
Fig. 1. Effect of in vitro treatments with varying concentrations of gar-
end of the experiment (day 15) 5 sh per aquaria (15 per lic extract on time to detachment and death of Gyrodactylus turnbulli.
treatment) were similarly examined. Infection prevalence Data points represent mean s.e.m. Different lower case letters indicate
and intensity were calculated as described above (Section signicant differences in time to detachment between treatments and dif-
2.5.2). ferent upper case letters indicate signicant differences in time to death
(one-way ANOVA with Tukeys post-hoc pairwise comparisons; p < 0.05).

2.7. Histopathology
Samples for histological examination were collected
from sh from the oral treatment trial on day 15. A total of
11 sh were sampled from the oral treatments and inde-
nite bath treatment and 9 sh from the control group. Fish
were euthanised and xed in 10% buffered formalin. Whole
xed sh were then cut transversally into approx. 0.5 cm
wide slices and processed using routine histological tech-
niques. Sections were then cut from the head, body and tail
portions and slides were stained with H&E and examined
under a light microscope.
2.8. Statistical analysis
All data were analysed by comparing treatments using
either a one-way ANOVA or a General Linear Model
(GLM) followed by Tukeys post-hoc pairwise compar-
isons (p < 0.05). Homogeneity of variance was tested
using Levenes test and normality was tested using the
AndersonDarling test. All percentage data were normal-
ized by arcsine square transformation prior to statistical
analyses to homogenise the variation and data are pre-
sented as back-transformed mean and upper and lower
95% condence limits. Cumulative mortality was ana-
lysed using the Log-Rank test. Signicance was accepted
when p < 0.05. All statistical analyses were performed using
Minitab 16.
This was followed by detachment of the parasite from
the host after which time the parasite continued to con-
tract, but with less frequency until death occurred. Time to
both detachment and death positively correlated to garlic
extract concentration; at 7.5 mL L1 garlic extract, para-
sites detached and died at 16.7 0.74 and 28.2 1.15 min,
respectively, whereas, at the highest concentration of
30 mL L1 , detachment and death occurred at 4.09 0.31
and 8.58 0.55 min, respectively (Fig. 1). Parasites in the
0 mL L1 control were observed for up to 1 h i.e. after the last
parasite had detached and died in the lowest aqueous garlic
extract concentration used i.e. 7.5 mL L1 , and no parasites
were observed to detach or die within this time period, sug-
gesting that results obtained for in vitro exposure to garlic
were not due to either host cell death encouraging parasite
detachment or as a result of the clove oil used to euthanise
the sh.
3.2. Experiment 2: Bath treatment of infected sh with
7.5 and 12.5 mL L1 of garlic extract
3.2.1. Toxicity test
Survival of guppies bathed in garlic extract was 100%
for up to 1 h at a concentration of 12.5 mL L1 aqueous
garlic extract (Supplementary material S1), therefore this
was chosen to be the maximum concentration for the bath
treatments.

3. Results
3.1. Experiment 1: In vitro parasite survival (7.5, 10,
12.5, 15, 20, 25, 30 mL L1 garlic extract)
A noticeable antagonistic effect was observed on gyro-
dactylid parasites attached to n clips following the
addition of garlic extract; parasites were initially observed
to move around rapidly, after which time they remained
in one place and started to contract and twitch violently.
3.2.2. In vivo bath treatments
Bathing in 7.5 and 12.5 mL L1 of garlic extract for
1 h signicantly (p < 0.05) reduced G. turnbulli infection
in guppies; mean intensity was 12.5 2.25 in the control
as compared to 1.33 0.88 and 0.3 0.3 in the 7.5 and
12.5 mL L1 treatments, respectively, and infection preva-
lence was 75% in the control as compared to 12.3% and
6.1% in the 7.5 and 12.5 mL L1 treatments, respectively
(Table 1).

Please cite this article in press as: Fridman, S., et al., Efcacy of garlic based treatments against monogenean parasites
infecting the guppy (Poecilia reticulata (Peters)). Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.002
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Table 1
Mean prevalence (%) and mean intensity s.e.m. of Gyrodactylus turnbulli on caudal, dorsal and pectoral ns of guppies following 1 h exposure to varying
concentrations of aqueous garlic extract (n = 3 replicates; 10 sh per replicate).

Aqueous garlic extract (mL L1 ) Before exposure After exposure

0 (control) 7.5 12.5 0 (control) 7.5 12.5

Mean prevalence (%) 80 80 66.7 75a 12.3b 6.1b

Mean intensity 12.6 0.83 11.6 2.99 14.4 2.24 12.5a 2.25 1.3b 0.88 0.3b 0.33

a, b, different letters within rows indicate signicant differences between treatments (one-way Anova with Tukeys post-hoc pairwise comparisons; p < 0.05).

Table 2
Effects of oral treatment with garlic on prevalence (%) and intensity of infection by G. turnbulli and Dactylogyrus sp. in the guppy. Percentage data are means
and 95% condence limits (appearing in brackets) of arcsine square transformed data, of 3 replicates (5 sh per replicate). Intensity data are means s.e.m.
of 3 replicates (5 sh per replicate).

Mean prevalence (%) Mean intensity

1 2
Total body Gills Tail1 Skin1 Gills2

Trial 1
Control 81.1a (9933) 70.8 (977)a 2.3 0.22 1.5 0.50a 1.5 0.26a
Praziquantal 27.9b (604) 0b 0.9 0.48 0.3 0.33a 0b
High garlic (4%) 53.3 (8618)ab 53.3 (6640)ab 1.9 0.48 1.9 0.89a 1.9 0.34a
Low garlic (2%) 75 (9816)a 46.7 (8113)ab 2.7 0.95 1.8 0.99a 1.7 0.44a
Trial 2
Control 80a (8080) 26.2 (3914)a 6.9 2.04a 6.1 2.79a 1.7 0.67
Fresh garlic 25 (750)ab 1.1 (91)c 1.7 0.44b 2 0b 1 0.57
High garlic (20%) 2.4 (192)b 2.4 (12)c 0.7 0.33b 0.7 0.33b 10
Low garlic (10%) 9.3 (330)b 13.9 (490.1)b 1.3 0.67b 0.8 0.4b 1.20.73

a, b, c, different letters within columns represent signicant differences between treatments (one-way ANOVA with Tukeys post-hoc pairwise comparisons;
p < 0.05).
1
Gyrodactylus turnbulli.
2
Dactylogyrus sp.

3.3. Experiment 3: Oral treatment of infected guppies to completely remove parasites from the gills (Table 2).
Infection intensity following Praziquantel treatment did
Microscopic examination of sh at the start of the trials not signicantly differ from the intensity in the control
revealed that body and ns were infected with G. turnbulli group on the body and ns, but was signicantly lower
and gills were infected with Dactylogyrus sp. We therefore than the control group on the gills (Table 2).
chose to analyse the effect of garlic on both parasites. Fish A signicant difference (p < 0.05) was observed in the
fed with garlic supplemented diets showed a signicantly cumulative mortality between the treatments and control
reduced mean prevalence (p < 0.05) of G. turnbulli infection in the second trial; cumulative mortality in the group fed
as compared to the control i.e. 10% and 20% were 9.3% and 20% was signicantly (p < 0.05) lower as compared to the
2.4%, respectively, as compared to 80% in the control but not group fed 10% garlic and the control (8.3%, respectively, as
at the lower supplemented doses i.e. 2% and 4% (Table 2). compared to 21.7% and 25%, respectively; Fig. 2).
Mean intensity of G. turnbulli was similarly signicantly
(p < 0.05) affected in the higher supplemented doses but
not at the, lower supplemented doses both in the tail i.e.
10% and 20% (1.3 0.67 and 0.7 0.33, respectively, as com-
pared to 6.9 2.04 in the control) and the skin (0.8 0.4
and 0.7 0.33, respectively, as compared to 6.1 2.79 in
the control) (Table 2). Dietary garlic similarly signicantly
reduced infection prevalence of dactylogyrids in sh gills
at 10% and 20% (13.9% and 2.4%, respectively, as compared
to 26.2% in the control) but did not signicantly affect the
mean intensity (Table 2).
Fresh crushed garlic used as an indenite bath in the
second trial similarly signicantly (p < 0.05) reduced para-
site prevalence both on the whole body (25%) and the gills
(1.1%) as compared to the control (Table 2). Correspond-
ingly, a signicantly lower mean intensity was observed
on the tail and skin of sh subjected to an indenite bath
of crushed garlic (6.9 and 6.1, respectively), but not on
the gills (Table 2). Praziquantal was seen to signicantly Fig. 2. Cumulative mortality (%) of guppies (Poecilia reticulata) from Trial
reduce parasite prevalence on the whole body (27.9%) and 2 fed with garlic supplemented diets or subjected to indenite bath of
fresh garlic for 14 days.

Please cite this article in press as: Fridman, S., et al., Efcacy of garlic based treatments against monogenean parasites
infecting the guppy (Poecilia reticulata (Peters)). Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.002
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3.4. Histopathology
Evidence of muscular dystrophy was observed in all
sampled sh. Ten out of 11 examined sh from the group
fed 20% garlic displayed evident dystrophy, which was
slight in 8 sh (about 3% of the muscle tissue seen in a sec-
tion was affected), and more severe in two cases (about 10%
of the muscle tissue). In the group fed 10% garlic 7 out of 11
sh displayed muscle dystrophy and in the continuous bath
treatment and control there was the least occurrence of
dystrophy, (2 out of 11 sh and 3 out of 9 sh, respectively;
data not shown).
4. Discussion
Research into the use of traditional, plant-based
medicines in the control and treatment of diseases in
shes, has increased. Cavallito and Bailey (1944) isolated
and identied the component of garlic responsible for the
antibacterial activity of crushed cloves, which was termed
allicin, a name derived from the latin name for garlic, A.
sativum. It was found to be a volatile, oxygenated sulphur
compound, scarcely miscible in water with the typical,
strong odour of freshly crushed garlic. It is formed upon
crushing of the garlic clove, whereupon its stable pre-
cursor alliin is converted to allicin by the action of the
enzyme alliinase. Allicin is a broad-spectrum antimicrobial
agent, exhibiting antibacterial, antifungal, antiprotozoal
and antiviral properties (Ankri and Mirelman, 1999).
The widespread and long standing use of garlic as a
food ingredient for humans suggests that garlic is non-
toxic to man (Bolton et al., 1982). In the present study
acute toxicity testing demonstrated that exposure of sh
to 12.5 mL L1 aqueous garlic extract for 1 h resulted in
no mortalities (100% survival), and in turn proved suc-
cessful in almost completely eradicating G. turnbulli in
the in vivo trials. It should be noted that the tolerance of
different sh species to compounds may vary consider-
ably therefore thorough testing of lethal concentrations
should be undertaken before a compound is used. The
mode of action of garlic on G. turnbulli is unclear. It has been
reported that Praziquantel acts on the membrane calcium
permeability of the body wall of the parasites resulting in
immediate muscular contraction, rapid vacuolisation and
disruption of the tegument and impairment of the attach-
ment mechanism (Redman et al., 1996). In this study the
parasites treated with aqueous garlic extract also appeared
to contract violently prior to detachment from the host
and continued to do so once detachment had occurred, up
until death. Interestingly Riad et al. (2009) describe various
ultrastructural alterations in Schistosoma mansoni worms
recovered from infected mice to which crude garlic juice
had been intragastrically administered for a period of 7
weeks; scanning electron microscopy revealed tegumental
abnormalities such as focal lesions, peeling and blebbing.
Transmission microscopy similarly revealed widespread
damage to tegument and sub-tegumental structures i.e.
oedema and vacuolisation.
In the present study determination of the garlic extracts
allicin content was not carried out. Militz et al. (2013a)
report allicin content of aqueous garlic extract, prepared
Please cite this article in press as: Fridman, S., et al., Efcacy of garlic based treatments against monogenean parasites
infecting the guppy (Poecilia reticulata (Peters)). Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.002
in the same way as this study i.e. a ratio of 10 g fresh gar-
lic per 50 mL, to be 0.76 0.03 L mL1 and this was not
found to decrease over the one month sampling time when
stored at 4 C. It may therefore be assumed that similarly
in the current work allicin levels remained stable over the
one month that the extract was stored; however, it should
be noted that allicin levels are likely to vary according to
garlic strain, seasonality and age (Lawson et al., 1991).
A noticeable antagonistic effect of aqueous garlic extract
was observed in the in vitro trials on G. turnbulli, which
resulted in detachment of the parasites and ultimately
death, and this was positively correlated to the concen-
tration of garlic applied. This effect of dose dependent
efcacy has similarly been reported by Militz et al. (2013a)
in trials testing the in vitro efcacy of aqueous garlic
extract against all life stages of the monogenean Neobene-
denia sp. infecting farmed barramundi (L. calcarifer) and
by Schelkle et al. (2013) in trials testing the in vitro ef-
ciency of various garlic compounds against G. turnbulli
in the guppy (P. reticulata). In agreement, in the current
study, aqueous garlic extract administered at 7.5 mL L1 ,
which is equivalent to 1500 mg fresh garlic L1 , was seen
to cause total detachment and death of G. turnbulli in vitro
within 40 min, however when administered at the high-
est concentration of 30 mL L1 , which is equivalent to
6000 mg fresh garlic L1 , total detachment and death of
parasites occurred within 16 min. It is important to note
that transmission of gyrodactylids occurs mainly during
direct contact between host sh, however detached spec-
imens can survive off the sh for several hours and can
reattach when they come into contact with a new host
(Cable and Harris, 2002). Therefore, parasite detachment
is not sufcient when testing the efcacy of a compound
and for a treatment to be effective death of the parasite
must also occur.
The current study complements prior work testing the
in vivo efcacy of garlic-based bath treatments against
gyrodactylids both in the guppy (P. reticulata) (Schelkle
et al., 2013) and the Nile tilapia (O. niloticus) (Abd El-Galil
and Aboelhadid, 2012). In the present work, bath expo-
sure to the lower concentration of 7.5 mL aqueous garlic L1
for 1 h (equivalent to 1500 mg fresh garlic L1 ) removed
most of the parasitic infection, reducing infection preva-
lence to 12.3% from the original 80%. It is possible that a
longer treatment period would result in complete clear-
ance of the infection. These previous studies and our data
suggest a concentration dependant efciency in removing
gyrodactylid infestation in vivo.
In a recent study Militz et al. (2013b) demonstrated the
efciency of dietary supplementation with garlic extract
in conferring resistance to post-treatment experimental
infection with the monogenean Neobenedenia sp. in the
barramundi (L. calcarifer). Both 50 and 150 mL garlic
extract/kg food reduced infection success, evidenced by
the lower infection prevalence and intensity when fed
for 30 days, but not when fed for 10 days. It appears
that by extending the in feed treatment period, complete
removal of the parasite may be achieved. Our study has
shown that, whilst garlic supplemented feed does not
completely remove all monogenean sp. after 14 days, it
does signicantly reduce infection levels and mortality
G Model
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S. Fridman et al. / Veterinary Parasitology xxx (2014) xxxxxx
rates. Infections with Gyrodactylus sp. are pathogenic
to shes principally at high infection levels (Cone and
Odense, 1984) therefore the benets of reducing parasite
loading are clear. Reduced labour time and related costs of
administering treatment in feed makes it a more practical
option than bath treatments. Recently there has been an
increase in aquaculture-related research reporting various
benecial effects of garlic in nsh culture including an
increase in stress resistance, growth promotion, appetite
stimulation, immunostimulation and antimicrobial prop-
erties (Colorni et al., 1998; Diab et al., 2002; Sahu et al.,
2007; Nya and Austin, 2009; Aly et al., 2010; Nya et al.,
2010; Vaseeharan et al., 2011; Guo et al., 2012; Talpur and
Ikhwanuddin, 2012).
Histological examination in the current study revealed
elevated muscle dystrophy in the sh fed diets supple-
mented with garlic powder as compared to the control.
Systemic toxic effects of dietary garlic have been described
in mammals suggesting that garlic may not be safe in all
doses; excess doses of garlic can cause toxicity, includ-
ing anemia and gastrointestinal problems in mammals
(Banerjee et al., 2003). However often histological changes
are seen to be dose dependent; Banerjee et al. (2001)
describe considerable cellular damage to both liver and
kidneys of rats following administration of fresh garlic
homogenate by gavage for 30 days at the higher dose of
1000 mg kg1 day1 but not at the lower doses i.e. 250
and 500 mg kg1 day1 . There is scant literature regard-
ing histological changes as a result of dietary garlic in
sh. Al-Salahy and Mahmoud (2003) administered raw
garlic juice at 2 g per kg body weight to the spiny cat-
sh (Chrysichthys auratus) and examined the effects on
histological structures of the liver and kidney; in sh
treated for both 5 and 11 days, livers are reported to show
cytoplasmic vacuolisation and hypertrophy of the hepato-
cytes due to fatty degeneration of lipid droplets leading to
the collapse of the blood sinusoids. Further study would
be required in order to examine more closely the neg-
ative pathological effect of garlic application in sh and
whether this pathology is reversible once application is
ceased.
To summarise, this study demonstrates the potential
of garlic in the treatment of monogenean infection in the
guppy both as an immersive treatment in vitro and in vivo
and as an in feed additive. Field studies are required to
assess potential of garlic in a full-scale aquaculture context.
Cost of using aqueous garlic extract at concentrations that
totally eradicate the problem may prove inhibitory but the
success of a compound may also be assessed by its ability
to simply reduce the parasite burden to manageable levels.
Parasites may exist in populations at low levels without
reaching epizootic levels therefore low dose treatments of
garlic may also prove to be effective management strategies
in the control of monogeneans.
Acknowledgements
The authors would like to thank Drs. Giuseppe Paladini
and Andy Shinn of the Parasitology Dept., Institute of
Please cite this article in press as: Fridman, S., et al., Efcacy of garlic based treatments against monogenean parasites
infecting the guppy (Poecilia reticulata (Peters)). Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.002
7
Aquaculture, University of Stirling, Scotland for identifying
the parasite Gyrodactylus turnbulli.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.vetpar.2014.02.002.
References
Abd El-Galil, M.A., Aboelhadid, S.M., 2012. Trials for the control of trichodi-
nosis and gyrodactylosis in hatchery reared Oreochromis niloticus fries
by using garlic. Vet. Parasitol. 185, 6273.
Al-Salahy, M.B., Mahmoud, A.A.B., 2003. Metabolic and histological stud-
ies on the effect of garlic administration on the carnivorous sh
Chrysichthys auratus. Egyptian Journal of Biology 5, 94107.
Aly, S.M., El Naggar, G.O., Mohamed, M.F., Mohamed, W.E., 2010. Effect of
garlic, echinacea, organic green and vet-yeast on survival, weight gain,
and bacterial challenge of overwintered Nile tilapia fry (Oreochromis
niloticus). J. Appl. Aquac. 22, 210215.
Ankri, S., Mirelman, D., 1999. Antimicrobial properties of allicin from gar-
lic. Microbes Infect. 2, 125129.
Bakke, T.A., Harris, P.D., Cable, J., 2002. Host specicity dynamics: obser-
vations on gyrodactylid monogeneans. Int. J. Parasitol. 32, 281308.
Banerjee, S.K., Maulik, M., Manchanda, S.C., Dinda, A.K., Das, T.K., Maulik,
S.K., 2001. Garlic-induced alteration in rat liver and kidney morphol-
ogy and associated changes in endogenous antioxidant status. Food
Chem. Toxicol. 39, 793879.
Banerjee, S.K., Sood, S., Dinda, A.K., Das, T.K., Maulik, S.K., 2003. Chronic
oral administration of raw garlic protects against isoproterenol-
induced myocardial necrosis in rat. Comp. Biochem. Physiol. C:
Toxicol. Pharmacol. 136, 377386.
Bolton, S., null, G., Troetel, W.M., 1982. The medicinal uses of garlicfact
and ction. J. Am. Pharmacol. Assoc. 22, 4043.
Buchmann, K., Jensen, P.B., Kruse, K.D., 2003. Effects of sodium percar-
bonate and garlic extract on Ichthyophthirius multiliis theronts and
tomocysts; in vitro experiments. North Am. J. Aq. 65, 2124.
Buchmann, K., Bresciani, J., Jappe, C., 2004. Effects of formalin treatment
on epithelial structure and mucous cell densities in rainbow trout,
Oncorhynchus mykiss (Walbaum), skin. J. Fish Dis. 27, 99104.
Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets
ecology on its own terms: Margolis et al. revisited. J. Parasitol., 83.
Cable, J., Harris, P.D., 2002. Gyrodactylid developmental biology: historical
review, current status and future trends. Int. J. Parasitol. 32, 255280.
Cable, J., van Oosterhout, C., 2007. The impact of parasites on the life his-
tory evolution of guppies (Poecilia reticulata): the effects of host size
on parasite virulence. Int. J. Parasitol. 37, 14491458.
Cavallito, C.J., Bailey, J.H., 1944. Allicin, the antibacterial principle of Allium
sativum. Isolation, physical properties and antibacterial action. J. Am.
Chem. Soc. 65, 19501951.
Colorni, A., Avtalion, R., Knibb, W., Berger, E., Colorni, B., Timan, B.,
1998. Histopathology of sea bass Dicentrarchus labrax experimentally
infected with Mycobacterium marinum and treated with streptomycin
and garlic Allium sativum extract. Aquaculture 160, 117.
Cone, D.K., Odense, P.H., 1984. Pathology of ve species of Gyrodactylus
Nordmann, 1832 (Monogenea). Can. J. Zool. 62, 10841088.
Diab, A.S., El-Nagar, G.O., Abd-El-hady, Y.M., 2002. Evaluation of Nigella
sativa L (black seeds; baraka), Allium sativum (garlic) and Biogen as
feed additives on growth performance and immunostimulants of O.
niloticus ngerlings. Suez Canal Vet. Med. J., 745775.
FAO, 2010. FishStat Plus 2010. Aquaculture Production Statistics:
19972008. Food and Agricultural Organisation of the United Nations,
Fisheries and Aqauculture Department, Rome, Italy.
Goven, B.A., Amend, D.F., 1982. Mebendazole/trichlorfon combination: a
new anthelmintic for removing monogenic trematodes from sh. Fish
Biol. 20, 373378.
Guo, J.J., Kuo, C.M., Chuang, Y.C., Hong, J.W., Chou, R.L., Chen, T.I., 2012.
The effects of garlic supplemented diets on antibacterial activity
against Streptococcus iniae and on growth in orange-spotted grouper,
Epinephelus coioides. Aquaculture 364, 3338.
Harris, P.D., Cable, J., Tinsley, R.C., Lazarus, C.M., 1999. Combined ribosomal
DNA and morphological analysis of individual gyrodactylid monoge-
neans. J. Parasitol. 85, 188191.
G Model
VETPAR-7117; No. of Pages 8 ARTICLE IN PRESS
8 S. Fridman et al. / Veterinary Parasitology xxx (2014) xxxxxx
Kildea, M., Allan, G., Kearney, R., 2004. Accumulation and clearance of the
anesthetics clove oil and Aqui-STM from the edible tissue of silver
perch (Bidyanus bidyanus). Aquaculture 232, 265277.
Lawson, L.D., Wood, S.G., Hughes, B.G., 1991. HPLC analysis of allicin
and other thiosuplhates in garlic clove homogenates. Planta Med. 57,
263270.
Lyles, A.M., 1990. Genetic variation and susceptibility to parasites: Poe-
cilia reticulata infected with Gyrodactylus turnbulli. In: Ph.D. Thesis.
Princeton University.
Militz, T.A., Southgate, P.C., Carton, A.G., Hutson, K.S., 2013a. Efcacy of
garlic (Allium sativum) extract applied as a therapeutic immersion
treatment for Neobenedenia sp. management in aquaculture. J. Fish
Dis., http://dx.doi.org/10.1111/jfd.12129.
Militz, T.A., Southgate, P.C., Carton, A.G., Hutson, K.S., 2013b.
Dietary supplementation of garlic (Allium sativum) to pre-
vent monogenean infection in aquaculture. Aquaculture,
http://dx.doi.org/10.1016/j.aquaculture.2013.05.027.
Millet, C.O.M., Lloyd, D., Williams, C.F., Williams, D., Evans, G., Saunders,
R.A., Cale, J., 2010. Effect of garlic and allium-derived products on the
growth and metabolism of Spironucleus vortens. Exp. Parasitol. 127,
490499.
Mitchum, D.L., 1995. Parasites of Fishes in Wyoming. Wyoming Game and
Fish Department Press, US, pp. 303.
Nya, E.J., Austin, B., 2009. Use of garlic, Allium sativum, to control
Aeromonas hydrophila infection in rainbow out, Oncorhynchus mykiss
(Walbaum). J. Fish Dis. 32, 963970.
Nya, E.J., Dawood, Z., Austin, B., 2010. The garlic component, allicin,
prevents disease caused by Aeromonas hydrophila in rainbow trout,
Oncorhynchus mykiss (Walbaum). J. Fish Dis. 33, 293970.
Paladini, G., Gustinelli, A., Fioravanti, M.L., Hansen, H., Shinn, A.P.,
2009. The rst report of Gyrodactylus salaris Malmberg, 1957 (Platy-
helminthes, Monogenea) on Italian cultured stocks of rainbow trout
(Oncorhynchus mykiss Walbaum). Vet. Parasitol. 165, 290297.
Paperna, I., 1991. Diseases caused by parasites in the aquaculture of warm
water sh. Annu. Rev. Fish Dis., 155194.
Pena, N., Auro, A., Sumano, H., 1988. A comparative trial of garlic, its
extract and ammoniumpotassium tartrate as anthelmintics in carp.
J. Ethnopharmacol. 24, 199203.
Please cite this article in press as: Fridman, S., et al., Efcacy of garlic based treatments against monogenean parasites
infecting the guppy (Poecilia reticulata (Peters)). Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.02.002
Redman, C.A., Robertson, A., Fallon, P.G., Modha, J., Kusel, J.R., Doenhoff,
M.J., Martin, R.J., 1996. Praziquantel: an urgent and exciting challenge.
Parasitol. Today 12, 1420.
Riad, N.H.A., Taha, H.A., Mahmoud, Y.I., 2009. Effects of garlic on albino
mice experimentally infected with Schistosoma mansoni: a parasito-
logical and ultrastructural study. Trop. Biomed. 26, 4050.
Sahu, S., Das, B.K., Mishra, B.K., Pradhan, J., Sarangi, N., 2007. Effects of
Allium sativum on the immunity and survival of Labeo rohita infected
with Aeromonas hydrophila. J. Appl. Ichthyol. 23, 8086.
Schmahl, G., Taraschewski, H., Mehlhorn, H., 1989. Chemotherapy of sh
parasites. Parasitol. Res. 75, 503511.
Schelkle, B., Shinn, A.P., Peeler, E., Cable, J., 2009. Treatment of gyrodactylid
infections in sh. Dis. Aquat. Org. 86, 6575.
Schelkle, B., Snellgrove, D., Cable, J., 2013. In vitro and in vivo efcacy of
garlic compounds against Gyrodactylus turnbulli infecting the guppy
(Poecilia reticulata). Vet. Par. 198, 96101.
Scott, M.E., Anderson, R.M., 1984. The population dynamics of Gyrodactylus
bullatarudis (Monogenea) within laboratory populations of the sh
host Poecilia reticulata. Parasitology 89, 159194.
Solenge, A., Bakke, T.A., Hansen, L.P., 1998. Potential for dispersal of Gyro-
dactylus salaris (Platyhelminthes, Monogenea) by sea-running stages
of the Atlantic salmon (Salmo salar): eld and laboratory studies. Can.
J. Fish. Aquat. Sci. 55, 507514.
Talpur, A.D., Ikhwanuddin, M., 2012. Dietary effects of garlic (Allium
sativum) on haemato-immunological parameters, survival, growth,
and disease resistance against Vibrio harveyi infection in Asian sea
bass, Lates calcarifer (Bloch). Aquaculture 364, 612.
Vaseeharan, B., Prasad, G.S., Ramasamy, P., Brennan, G., 2011. Antibacterial
activity of Allium sativum against multidrug-resistant Vibrio harveyi
isolated from black gill diseased Fenneropenaeus indicus. Aquac. Inter.
19, 531539.
Williams, C.F., Lloyd, D., Kolarich, D., Alagesan, K., Duchne, M., Cable,
J., Williams, D., Leitsh, D., 2012. Disrupted intracellular redox
balance of the diplomonad sh parasite Spironucleus vortens by 5-
nitroimidazoles and garlic-derived compounds. Vet. Parasitol 190,
6273.