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Research Article
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In vitro Effect of Garlic Extract and Metronidazole Against Neoparamoeba pemaquidensis, Page 1987 and Isolated Amoebae from Atlantic Salmon |
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R. Peyghan,
M.D. Powell
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M.R. Zadkarami
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ABSTRACT
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Neoparamoeba pemaquidensis believed to be the most prevalent parasite of Atlantic salmon industry in Australia. In the present study, the in vitro effects of crude extract of garlic and metronidazole were investigated using a primary culture toxicity assay. Garlic extract appeared to be completely effective at killing a cultured strain (NP251002) of Neoparamoeba pemaquidensis in vitro at a dilution of 1:100 with in 24 h. The number of viable Amoebae after using garlic extract in lower dilutions (1:200, 1:400, 1:800, 1:1000) for 24 h, also were significantly lower than in the control group. Garlic extract was also efficacious at killing wild type Amoebae that isolated from the diseased fish showing clinical signs of AGD. Metronidazole had no clear effect against Neoparamoeba pemaquidensis (NP251002) even in a concentration of 50 mg L-1 for 24 h. However some morphological changes have occurred in metronidazole-treated Amoebae after 5 days of exposure.
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INTRODUCTION
Amoebic Gill Disease (AGD) is an important disease of
Atlantic salmon (Salmo salar) in Tasmania and has been reported
from other areas (Munday et al., 2001). The morphology of Neoparamoeba
pemaquidensis, the causative agent of AGD, has been extensively studied
and a new species N. branchiophila has also been identified in
AGD infections (Dykova et al., 2005). Typical pathological changes
of fish infected with Neoparamoeba pemaquidensis are the hypertrophy
and desquamation of surface epithelial cells within the immediate vicinity
of attachment, hyperplasia and thickening of secondary lamellae as well
as edema of the epithelium. The fishes are highly susceptible to infection
in the sea water and the resulting gill damage and its influence on respiratory
and acid-base balance contributes considerably to the lethal effect caused
by the parasite (Powell et al., 2000; Powell
and Nowak, 2003; Adams and Nowak, 2003).
There are several reports about the treatment of AGD
affected fishes (Zilberg et al., 2000; Parsons et al., 2001;
Clark et al., 2003; Roberts and Powell, 2004; Harris et al.,
2004) but, the only effective commercial scale treatment for AGD to date
is freshwater bathing that can be effective in the removal of majority
of Neoparamoeba sp. from the gills of the affected fishes (Parsons
et al., 2001). However, there are some reports about the effectiveness
of other compounds as either bath treatments or as potential in feed amoebocides
(Powell et al., 2003; Powell and Clark, 2003; Powell et al.,
2005). Freshwater bathing to successfully avoid AGD is a critically important
industry concern in term of production costs. Therefore the development
of more effective drugs to control AGD in cultured fish is being sought.
Garlic (Allium sativum) is known as a potent medicine
with broad therapeutic properties ranging from antibacterial to anticancer,
antiparasitic and anticoagulant (Harris et al., 2001a; Ankri and
Mirelman, 2001). Crude extracts of garlic and many other plants have been
used as chemotherapeutics in different investigations (Emeruwa, 1982;
Tona et al., 1998; Chinoy et al., 1995; Udoh and Kehinde,
1999; Asres et al., 2001; Buchmann et al., 2003; Ijah and
Oyebanji, 2003; Lawson, 1996). The freshly cut slices of garlic bulb have
sulfur-containing compounds such as allicin, the primary contributor of
the pungency and medicinal properties of garlic (Ankri and Mirelman, 2001).
Whole garlic typically contains about 1% allein, which is the odorless
precursor of allicin. Allinase, which is an enzyme that is located within
a separate compartment in natural garlic, converts allein into the biologically
active allicin following the crushing of garlic and the contact between
the enzyme and the substrate (Langa et al., 2004). Purification
of allicin is a difficult tusk and was done by differential adsorption
of the reaction components on a neutral polystyrene resin (Miron et
al., 2004).
Other important sulphur-containing compounds present
in garlic homogenate are allyl methyl thiosulphonate, 1-propenyl allyl
thiosulphonate and L-glutamyl-S-alkyl-L-cysteine. Garlic oil consists
of the diallyl (57%), allyl methyl (37%) and dimethyl (6%) mono to hexa
sulphides. A typical commercial preparation of garlic oil contains 26%
diallyl disulphide (DADS), 19% diallyl trisulphide (DATS), 15% allyl methyl
trisulphide, 13% allyl methyl disulphide, 8% diallyl tetrasulphide, 6%
allyl methyl tetrasulphide, 3% dimethyl trisulphide, 4% penta sulphide
and 1% hexa sulphide. All polysulphides show antioxidant effects in
vitro but this has yet to be confirmed in vivo. Diallyl polysulphide
(S = 3 to 7) inhibits the formation of thiobarbituric acid reactive substances
in vitro (Horie et al., 1992). Quantitative tests revealed
that there were 17.8 n moles sulfhydryl (SH)/ml of 40 KD garlic protein
localized in the parenchyma sheath cells and the cortical cells of garlic
bulbs (Wen et al., 2004).
Metronidazole is the only 5-nitroimidazole approved for
used in the United States and it is the drug of choice for treating giardiasis
and amoebiasis (mainly referring to disease caused by Entamoeba histolytica)
in many countries (Campanati and Monteiro-Leal, 2000). Both giardiasis
and amoebiasis causative agents are anaerobic endoparasites and act differently
from Neoparamoeba sp. which is an aerobic ectoparasite, but this
parasite might also respond to metronidazole treatment.
In this study, the anti-amoebic effect of garlic extract
as a natural extract and metronidazole as a potentially amoebostatic and
anti-protozoal drugs were examined for their efficacy and toxicity to
Neoparamoeba sp. in vitro.
MATERIALS AND METHODS
This study used Neoparamoeba pemaquidensis (NP251002)
and amoeba isolated from Atlantic salmon, originated from one of Tasmanian
hatcheries which were introduced at the Aqua center of school of Aquaculture,
University of Tasmania, Launceston-Australia. The laboratory examinations
and toxicity tests were done from October 2004 to January 2005.
Garlic extract preparation: Raw garlic homogenate prepared by
crushing the 10 g garlic bulb in 100 mL water (water temperature: 20-22°C)
in a pestle and mortar and passing the homogenate through paper filter
and antibacterial paper (0.2 μm filter). For preparation of heated
garlic extract, after the filtration of the extract, it placed in 80°C
oven for 2 min. As a result of this heating, the transparent garlic extract
changed to cloudy white. Forty microliter per milliliter of an antibiotic
mixture: (Streptomycin 0.1 g, penicillin 0.1 g, carbenicillin 0.1 g, ampicillin
0.25 g, distilled water 10 mL) (Sigma, St. Louis, MO, USA) was added to
the garlic extract and the control groups wells.
In vitro toxicity test with Neoparamoeba pemaquidensis
(NP251002) Neoparamoeba pemaquidensis preparation: Neoparamoeba
pemaquidensis (strain NP251002) were cultured on seawater malt yeast
extract (Oxoid, Victoria, Australia) agar plates at 18°C. Amoebae
were not fed additional bacterial cultures during the culture period.
Amoebae were harvested from the agar plate by washing
and suspending them in 0.2 μm filtered seawater. The harvested Amoebae
were counted using a haemocytometer (Neubauer, BS 748) and their viability
assessed using trypan blue exclusion staining (Sigma) according Powell
et al. (2003). The trypan blue staining was used for assaying the
live Amoebae (live Amoebae do not taking up the trypan blue stain).
Fresh garlic extract toxicity test: Two hundred microlitres of
cultured Amoebae solution was added to each well in a 96 well micro plate
(Sarstedt-Australia). Twenty microlitres of garlic extract was added to
each experimental group to make a final dilution of 1:100. By adding water,
the serial dilution of 1:200, 1:400, 1:800, 1:1000, 1:1200 and 1:1400
were prepared and finally all of the amoeba solutions were incubated at
18.5°C for 24 h.
In other experiment, 200 μL of cultured Amoebae
solution was added to each well in a 96 well micro plate (Sarstedt-Australia).
Twenty microlitres of garlic extract was added to each experimental group
to make a final dilution of 1:100. Dilution was incubated at 18.5°C
for 8 h and amoeba number of each well were counted at 2 h intervals.
Heated garlic extract toxicity test: For preparation of heated
garlic extract, after the filtration of the extract, it placed in 80°C
oven for 2 min 200 μL of cultured Amoebae solution was added to each
well in a 96 well micro plate (Sarstedt-Australia). Twenty microliter
of the extract was added to each experimental group to make a final dilution
of 1:100. By adding water, the serial dilution of 1:200, 1:400, 1:800
and 1:1000 were prepared and finally all of the amoeba solutions were
incubated at 18.5°C for 24 h.
In vitro toxicity test with wild-type Amoebae Wild type Amoebae
isolation: In order to confirm the efficacy of garlic extract on wild
type of amoebae, Amoebae isolation from AGD-affected Atlantic salmon from
an ongoing laboratory infection at University of Tasmania was conducted.
Amoeba isolation from diseased fish was carried out according to Morrison
et al. (2004).
Fresh garlic extract toxicity test: Two hundred microlitres of
cultured Amoebae solution was added to each well in a 96 well micro plate
(Sarstedt-Australia). Twenty microlitres of garlic extract was added to
each experimental group to make a final dilution of 1:100. By adding water,
the serial dilution of 1:200, 1:400, 1:800 and 1:1000 were prepared and
finally all of the Aamoeba solutions were incubated at 18.5°C for
24 h.
In other experiment, 200 μL of isolated Amoebae
solution was added to each well in a 96 well micro plate (Sarstedt-Australia).
Twenty microlitres of garlic extract was added to each experimental group
to make a final dilution of 1:100. Dilution was incubated at 18.5°C
for 8 h and amoeba number of each well were counted at 2 h intervals.
Metronidazole toxicity test: Using same method, 200 μL of
amoeba suspension was continuously exposed to metronidazole (Sigma, St.
Louis, MO, USA) in different concentrations (0.1-100 mg L-1)
over 6 days. The number of Amoebae in all experimental and control groups
was determined using a haemocytometer (Neubauer), after staining with
0.5% trypan blue-seawater mixture at a dilution of 1:5 (Powell et al.,
2003). Six replicate counts were made with counting of nine large haemocytometer
squares per replicate.
Light microscopic observation of amoebae: One hundred microlitres
of amoeba suspensions from garlic exposed Amoebae (from 1:100 dilution)
and metronidazole (from concentration of 100 mg L-1) exposed
Amoebae was placed on to a glass slide for 1 h for adherence of amoebae.
Non-adherence cells were washed off. The slides were fixed by 2 drops
of methanol. After drying in room temperature, the slides were stained
by 1:10 dilution of giemsa and methylene blue stain for 15 min. A Leica
DC300F digital camera (Leica-Germany) mounted to a light microscope (Olympus)
was used for image capture.
Statistical analysis: The data was transformed to the Log-units
to see the real effect and was analyzed by one-way (data by groups) (ANOVA).
Post hoc Tukey`s, alpha (0.05) test was used for mean comparisons where
the difference was significant between the groups. Statistics for each
analysis were based on cases with no missing data for any variable in
the analysis. SPSS (Version 10.0, SPSS Science) were used for data analysis
and presentation. Data are presented as mean±standard error of
mean (SE).
RESULTS
Fresh garlic extract toxicity test:
A: Neoparamoeba, NP251002: In the 24 h toxicity test, the Amoebae
count for 1:100 and 1:200 dilutions was zero and in the 1:400, 1:800 and
1:1000 dilution of garlic solution was significantly lower then the control.
At higher dilutions (1:1200 and 1:14000) there were no significant differences
between garlic-exposed groups and the control (Fig. 1).
Numbers of live Amoebae (Neoparamoeba, Np251002) in the experimental
group treated with garlic extract (dilution of 1:100) began to decrease
significantly after 2 h of exposure and reached to zero after 8 h (Fig.
2).
Wild type Amoeba: The wild type Amoebae number count in the experimental
group treated with 1:100 garlic extract also decreased significantly (Fig.
3). However, in other
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Fig 1: |
In vitro toxicity
test of Neoparamoba pemaquidensis (cultured strain NP251002)
to various dilutions of fresh garlic extract in 24 h |
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Fig 2: |
In vitro toxicity
test of Neoparamoba pemaquidensis (cultured strain NP251002)
exposed to 1:100 dilution of fresh garlic extract in 8 h |
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Fig 3: |
In vitro
toxicity test of wild type amoeba to various dilutions of fresh garlic
extract in 24 h |
dilutions (1:400, 1:800 and 1:1000) there was not any significant difference
between the experimental groups and the control. Numbers of live Amoebae
(wild type) in the experimental group treated with garlic extract (dilution
of 1:100) began to decrease significantly after 6 h of exposure (Fig.
4).
Heated garlic extract toxicity test:
In the toxicity test with heated garlic extract, the Amoebae count (Neoparamoeba,
NP251002) in the control group significantly higher than the 1:100 dilution
after 24 h exposure to heated garlic extract. However, at higher dilutions
(1:400, 1:800 and 1:1000) garlic had not any significant effect in decreasing
Amoebae count in 24 h (Fig. 5).
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Fig 4: |
In vitro toxicity
test of wild type Amoeba exposed to 1:100 dilution of fresh garlic
extract in 8 h |
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Fig 5: |
In vitro toxicity
test of Neoparamoba pemaquidensis (cultured strain NP251002)
exposed to 1:100 dilution of heated garlic extract over 24 h |
Metronidazole toxicity test: Metronidazole had no significant
effect against Neoparamoeba in a concentration of lower than 50 mg L-1
in 24 h. The Amoebae count, however, in the control group was significantly
higher than the 100 mg L-1 group after 6 days of exposure (Fig.
6).
Morphological changes: Garlic extract caused cell vacuolization
and swelling that lead to the rapture and death of the amoebae. These
changes were not seen in Amoebae of the control groups. In comparison
with control group amoebae, some morphological changes
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Fig 6: |
In vitro toxicity
test of Neoparamoba pemaquidensis (cultured strain NP251002)
exposed to metronidazole in different concentrations in 6 days |
(abnormality of cell wall and the cytoplasm) in treated
Amoebae were observed after 5 days exposure to metronidazole.
DISCUSSION
In this study garlic extract had an obvious antiamoebic
effect. However, an effective treatment using natural sources would be
less expensive then some other synthetic drugs. Moreover, this kind of
product may be safe for fish and have less impact on the environment.
Garlic (Allium sativum) has played an important medicinal role
for centuries. Raw garlic homogenate is the major preparation of garlic
that has been subjected to intensive scientific study. Allicin is the
major thiosulphinate compound found in garlic homogenate (Benerjee et
al., 2003). It is the one of the active principles of freshly crushed
garlic homogenates that was found to exhibit antibacterial and antiparasitic
activity by means of chemical reaction with thiol groups of various enzymes
(Ankri and Mirelman, 2001). Inhibito-lethal activity against Giardia
intestinalis was noted with crude extract of garlic at 25 μg
mL-1 and lethal dosage was established as 50 μg mL-1
(Harris et al., 2001b).
In this study metronidazole had not any significant effect
on Neoparamoeba sp. in concentration lower then 50 mg L-1.
Metronidazole is used to treat a broad range of infections caused by anaerobic
protists and bacteria. This drug is also effective against human amoebiasis.
The selective cytotoxicity of metronidazole relies on biochemical properties
of anaerobic organisms that are lacking in the aerobic cells. For aerobic
cells the inhibitory concentration of metronidazole is two to three orders
of magnitude higher than that for anaerobes (Land and Johnson, 1997).
Some ultra structural studies are needed for further determination of
morphological changes (abnormality of cell wall and the cytoplasm) of
the Amoebae that were observed after treatment. Morphological changes
of metronidazole may be the effect of DNA changes in the treated amoebae.
Metronidazole appeared to have a role in inducing DNA damage and DNA breakage
due to treatment in human lymphocytes through the futile cycle (Menendez
et al., 2001; Re et al., 1997). However because the morphological
changes in our study were not characterized by more specific staining
and or ultra structural studies, further studies in this respect is recommended.
Garlic and metronidazole have not previously been investigated
against N. pemaquidensis. This study showed that garlic extract
strongly influenced the survival of the gill Amoebae in vitro.
In the present investigation, it is showed clearly that the concentration
of 100 mg L-1 is specifically important for effecting survival
of N. pemaquidensis in 1 h. Although N. pemaquidensis can
withstand relatively metronidazole, it is showed that higher concentration
of metronidazole (100 mg L-1) kills the majority of Neoparamoebae
causing AGD in salmon in vitro. Previous research has suggested
that only fresh water can kill N. pemaquidensis (Clark, 2002).
There are a lot of risk factors within the Tasmanian salmon aquaculture
industry when fresh water are using for treatment. Bath duration typically
ranges from 2 to 4 h, the water used to bathe fish is often hard and not
all of the Amoebae are removed from the gills. Survival of some Amoebae
on the gills during bathing either within the structure of the gill (Parsons
et al., 2001) or in the gill mucus (Clark, 2002) would mean that
freshwater resistant Amoebae may be developed. Other chemical treatments
were variable in their ability to reduce the relative survival of gill
Amoebae including N. pemaquidensis in vitro. Chloramine-T
and hydrogen peroxide showed the most promise. Chlorine-based disinfectants
such as chloramine-T primarily act by the release of hypochlorite (Booth
and McDonald, 1988). The effects of these disinfectants on the Amoebae
may be mitigated by the presence of mucus. This may be particularly important
when using disinfectants to remove and kill Amoebae on the gills of fish.
In the latter case, hypochlorite results in irritation and alteration
in gill epithelial membrane permeability (Powell and Perry, 1998) resulting
in a hyper secretion of bronchial mucus, potentially aiding the removal
of the parasites.
In conclusion, we have demonstrated that, fresh garlic
extract in dilution of 1:100 (concentration of about 10 g L-1)
in vitro, is efficacious in killing the Neoparamoebae sp.
after 8 h exposure in vitro. Therefore garlic extract or
its active antimicrobial agent (Allicin) may be used alone or in combination
with other effective drugs (or fresh water bath) in the treatment of amoebic
gill disease in farmed Atlantic salmon. However, before that it is necessary
to study the toxicity and pathological effect of garlic extract on Atlantic
salmon and establish the efficacy of garlic extract for the removal of
Neoparamoeba sp. from the gills of infected fish.
ACKNOWLEDGMENTS
We would like to thanks the contributions of Dr. P. Crosbie
for supplying culture Amoebae (NP251002), some isolated amoeba and the
antibiotics mixture. This work formed part of a project of Aquafin CRC
and received funds from the Australian Government`s CRCs Program, the
Fisheries R and D Corporation and other CRC Participants and was done
as part of sabbatical study of first author funded by Shahid Chamran University,
Ahvaz- Iran.
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