Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CARVACROL AND/OR THYMOL OR COMPOSITION THEREOF FOR PREVENTING OF INFECTION OR
INFESTATION OF AN ECTOPARASITIC COPEPOD IN FISH.
Field of the invention
The present invention relates to a carvacrol and/or thymol or a composition
comprising one or both of the active compounds, and to a method of killing,
combating or controlling sea lice in fish. The present invention further
relates to the
use Carvacrol and/or thymol for manufacture of a pharmaceutical composition
and to
a feed for fish.
Background of the invention
Infectious parasitic disease breakouts are a serious problem in aquaculture of
various species. Economical losses caused by these diseases can be tremendous
and significant research effort has been dedicated to treat and prevent these
diseases in aquaculture production. Moreover, parasitic infestations in farmed
aquatic animals are not only harmful for the cultivated animals; they also
represent a
constant and severe thread for wild populations, which can be infected by the
farmed
animals. This is another reason why an effective control and treatment of
parasites
and other infectious diseases is of great importance from an environmental
point of
view.
Serious disease outbreaks caused by ectoparasitic sea lice represent an
increasing
problem in salmon production in countries such as Norway, Chile, Scotland,
Ireland
and Canada over many years and until today. Infestations of farmed fish,
especially
of the Atlantic salmon Salm salar by the ectoparasitic copepods
Lepeophtheirus
salmonis, Caligus elongatus and Caligus rogercresseyi are not only harmful to
the
fish, they may eventually also lead to increased mortality in serious cases of
infections.
Sea lice (e.g. Caligus rogercresseyi and L. salmonis) infestation is for the
time being
one of the most important health challenges for the salmon production
industry. This
external parasite impairs production efficiency, causes stress and damages to
the
fish and is as a secondary infection a possible vector for other pathogens
like the
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salmon alpha virus (for Pancrease Disease), Infectious Salmon Anaemia virus
(ISAv)
and the Piscirickettsia salmonis. In Chile, like in many other countries
producing
Salmonids, infestation rates have increased significantly mainly affecting
species like
the Atlantic salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and
Coho
salmon (Oncorhynchus kisutch). In Norway, sea lice infections are considered
as
one of the most serious health problems in aquaculture of Salmon. This higher
infestation rate is probably a consequence of a combination of factors
including the
development of extensive resistances of the sea lice to most of the applied
chemical
antiparasite agents used in treatment today.
Commonly applied chemotherapeutic agents for the treatment of sea lice
infections
are e.g. belonging to the group of organophosphates, pyrethroids, hydrogen
peroxides, chitin synthesis inhibitors and emamectin. However, resistances,
often
also including multiple and cross-resistances, of sea lice often result in
limited
success when applying many of the above-mentioned commonly applied anti-sea
lice compounds.
The toxicity of many pharmacological effective compounds for the treated
animals as
wells as a limited biological degradation are limiting their application for
aquaculture
purposes. Compounds should preferably be non-toxic for the fish in the applied
concentrations and due to environmental issues, the compounds should be fast
and
easily degradable in the natural environment without any unfavorable and
harmful
accumulation in the nutritional chain. When supplied with the diet, compounds
should either be degraded or excreted by the organism after a reasonable time
period. This is especially important since farmed animals will eventually be
consumed by humans.
Therefore it is very important to find new compounds and ways to treat and
prevent
ectoparasites infections in fish, especially of sea lice.
The objective technical problem of the present invention is to provide new
effective
agents and methods for killing copepod ecto-parasites, and/or to inhibit the
infestation of ectoparasites in fish, such as sea lice and other copepod
parasites.
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Summary of the invention
A first aspect of the present invention relates to carvacrol and/or thymol or
a
composition comprising one or both of the active compounds for use in the
treatment
and/or prophylaxis of ectoparasitic copepod infection and/or infestation in a
fish.
Preferable, the ectoparasitic copepod is a sea lice.
Preferable, the sea lice is selected from Lepeophtheirus salmonis, Caligus
elongatus
and Caligus rogercresseyL
Preferable, the ratio of carvacrol:thymol is in the range of 1:3 to 3:1, more
preferable
of 1:2 to 2:1, most preferred about 1:1 based on weight.
Preferable, said fish is a Salmonidae and more preferred a fish selected from
the
group consisting of Atlantic salmon (Salmo salar), Rainbow trout
(Onchorhynkuss
mykiss), Coho salmon (Oncorhynchus kisutch) and Artic charr (Salvelinus
alpinus).
Preferably, the active compounds are used for reduction of the total number of
adult
sea lice stages infested or re-infested on fish.
Preferable, carvacrol and/or thymol is added to a feed composition in a total
concentration between 0.001% to 0.8% of the feed, preferably 0.003 % to 0.6 %
and
more preferred between 0.003 and 0.012 wt % of the feed.
Preferable, the total concentration of carvacrol and/or thymol is 30 mg/kg or
more,
preferably 45 mg/kg feed or more, more preferred at least 100 mg/kg and most
preferred at least 120 mg/kg feed.
Preferable, carvacrol and thymol is comprised in a ratio of 1:1.
Preferable, the feed comprises other conventional feed ingredients in the fish
feed.
Preferably, the feed further comprises one or several of the following
ingredients
selected from the group of nucleotides, prebiotics, vitamin C and immune
stimulants,
which can be specific or non-specific.
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Preferable, the compound or composition is supplied to fish for at least 7
days, more
preferred for at least 21 days and most preferred for at least 28 days.
Preferable, the treatment with the active compounds is combined with other
chemical
or medical agents against sea lice.
Preferable, the active compounds are specifically used to combat certain life
stages
of sea lice.
Preferable, the active compounds are used to reduce the infestation of gravid
female
sea lice on the fish.
Preferable, the fish are fed continuously the feed comprising the active
compounds
as part of their daily diet.
Preferable, the active compound or compounds are applied in form of a water
bath
treatment.
A second aspect of the present invention relates to a method of killing,
combating or
controlling ectoparasitic copepods, wherein the method comprising applying
carvacrol and/or thymol or a composition comprising one or both of the active
compounds in order to kill the sea lice, make them lethargic or prevent
infestation to
fish.
Preferable, the ectoparasitic copepod is a sea lice.
Preferable, the sea lice is Lepeophtheirus salmonis, Caligus elongatus or
Caligus
rogercresseyL
Preferable, said fish is a Salmonidae, preferably selected from the group
consisting
of Atlantic salmon (Salmo salar), Rainbow trout (Onchorhynkuss mykiss), Coho
salmon (Oncorhynchus kisutch) and Artic charr (Salvelinus alpinus).
Preferable, said active compound, mixture of compounds or composition is
applied
to a locus to be protected from the parasites.
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Preferable, the locus is an aquatic environment and wherein the active
compound or
composition comprising the active compound is applied directly to the aquatic
locus.
Preferable, said locus contains fish and wherein the sea lice are located on
said fish
5 .. or said fish is susceptible for infestation or infection of said sea
lice, wherein the
active compound or mixture of compounds is applied directly to the fish,
preferable
by topical or oral application.
Preferable, the active compound or mixture of compounds are applied to the
fish as
a component of the feed fed to the fish.
Preferably, the ratio of carvacrol:thymol is in the range of 1:3 to 3:1, more
preferable
1:2 to 2:1, and most preferable about 1:1, based on weight.
Preferably, one or more of the active compounds or a composition comprising
the
active compounds are supplied to fish as part of feed for at least 7 days,
more
preferred for at least 21 days and most preferred for at least 28 days.
A third aspect of the present invention relates to a use of carvacrol and/or
thymol for
manufacture of a pharmaceutical composition for the treatment and/or
prophylaxis of
ectoparasitic copepod infection and/or infestation in a fish.
Preferably, the ectoparasitic copepod is a sea lice.
Preferably, the sea lice is selected from Lepeophtheirus salmonis, Caligus
elongatus
and Caligus rogercresseyi.
Preferably, the ratio of carvacrol:thymol is in the range of 1:3 to 3:1, more
preferable
of 1:2 to 2:1, most preferred about 1:1 based on weight.
Preferably, said fish is a Salmonidae.
More preferably, said fish is selected from the group consisting of Atlantic
salmon
(Salmo salar), Rainbow trout (Onchorhynkuss mykiss), Coho salmon (Oncorhynchus
kisutch) and Artic charr (Salvelinus alpinus).
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Preferably, the pharmaceutical composition is for a reduction of the total
number of
adult sea lice stages infested or re-infested on fish.
Preferably, the pharmaceutical composition comprising carvacrol and/or thymol
is
added to a feed composition in a total concentration of carvacrol and/or
thymol from
0.001% to 0.8% of the feed, preferably from 0.003 % to 0.6 % and more
preferred
from 0.003 and 0.012 wt % of the feed.
Preferably, the pharmaceutical composition comprising carvacrol and/or thymol
is
added to a feed composition, which has a total concentration of carvacrol
and/or
thymol of at least 30 mg/kg feed, preferably at least 45 mg/kg feed, more
preferred at
least 100 mg/kg and most preferred at least 120 mg/kg feed.
Preferably, carvacrol and thymol is comprised in a ratio of 1:1.
Preferably, the feed comprises other conventional feed ingredients in the fish
feed.
Preferably, the feed further comprises one or several of the following
ingredients
selected from the group of nucleotides, prebiotics, vitamin C and immune
stimulants,
which can be specific or non-specific.
Preferably, the compound or composition is supplied to fish for at least 7
days, more
preferred for at least 21 days and most preferred for at least 28 days.
.. Preferably, the treatment with the active compounds is combined with other
chemical
or medical agents against sea lice.
Preferably, the active compounds are specifically used to combat certain life
stages
of sea lice.
Preferably, the active compounds are used to reduce the infestation of gravid
female
sea lice on the fish.
Preferably, the fish are fed continuously the feed comprising the active
compounds
as part of their daily feed.
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Preferably, the pharmaceutical composition is applied in form of a water bath
treatment.
In a fourth aspect the present invention relates to a feed for fish comprising
carvacrol
and/or thymol in a total concentration of 0.001% to 0.8% of the feed,
preferably
0.003 % to 0.6 % and more preferred between 0.003 and 0.012 wt A of the feed.
Preferably, the total concentration of carvacrol and/or thymol is at least 30
mg/kg,
preferably at least 45 mg/kg feed, more preferred at least 100 mg/kg and most
preferred at least 120 mg/kg feed.
Preferably, the ratio of carvacrol:thymol is in the range of 1:3 to 3:1, more
preferable
of 1:2 to 2:1, most preferred about 1:1 based on weight
Brief Description of the Drawings
=
Embodiments of the invention will now be described, by the way of examples
with
reference to the following diagrams, wherein;
Figure 1 shows the observed mortality of copepodids of C. rogercresseyi (as
proportion of the number of lice at the start of the study), over a period of
48 h at
different doses of Carvacrol. Each panel represents a time point (zero to 48
hours)
from the start of the study.
Figure 2 shows the percentage mortality of copepodids of C. rogercresseyi
exposed
to different doses of Carvacrol (pUL) during 48 hours. The error bars
represent the
standard deviation obtained for the replicas in each dose of Carvacrol.
Figure 3 shows the observed mortality of adults of C. rogercresseyi (as
proportion of
initial), over a period of 48 h at different doses of Carvacrol.
Figure 4 shows the percentage mortality of adults of C. rogercresseyi exposed
to
different doses of Carvacrol (pL/L) during 48 hours. The error bars represent
the
.. standard deviation obtained for the replicas in each dose of Carvacrol.
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Figure 5 shows the proportion ( /0) of reduction in expected sea lice (C.
rogercresseyi) infested over the fish when fed two doses of Carvacrol +Thymol
in
feed in comparison to the control feed.
Figure 6 shows (for example 4) feed consumed by the fish in each treatment
during
the trial. a) Specific Feeding Rate (SFR) for fish fed with high dose of
Carvacrol, b)
SFR for fish fed with low dose of Carvacrol and c) SFR for fish fed with
control feed.
Figure 7 shows (for example 4) sea lice (C. rogercresseyi) infestation
expressed as
total number of lice per tank which contained 35 fish , where dot is the
average
infestation and lines indicate credible intervals (95%) in each dose (low and
high)
and control.
Figure 8 shows expected average Caligus counts over whole monitoring period
(posterior median) for each life stage and treatment with 95% credible
intervals (error
bar) (example 5, experiment 1).
Figure 9 shows expected Caligus reinfestation counts (posterior median, solid
line)
for juveniles, adults and total lice by each treatment with 95% credible
intervals
(shadowed area, shown only for 0 and 120 ppm). Black line indicates control
with 0
ppm (example 5, experiment 2).
Figure 10 shows expected Caligus reinfestation counts for gravid females,
females
and adults for each treatment with 95% credible intervals (shadowed area).
Black
line means control (example 5, experiment 2).
Figure 11 shows linear and quadratic coefficients of the dose-response model
with
95% credible intervals for each sampling day (example 5, experiment 2). The
quadratic effects are magnified (x100) to make them visible in the same plot
as the
linear terms. See text for explanation.
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Detailed Description
Example 1 - Effect of Carvacrol on copepodids of Calicius rocercressevi
We have tested the compound Carvacrol against copepodids of Caligus
rogercresseyi by an in vitro method.
The objectives of this study is to i) determine if Carvacrol induces mortality
of
copepodids of Caligus rogercresseyi, and ii) determine the LD50 (Lethal Dosis
that
causes 50% mortality) of Carvacrol against copepodids of Caligus rogercresseyi
in
vitro.
Materials and Methods
During January 2009 a study was conducted to determine the LD50 of Carvacrol
against copepodids stage of Caligus rogercresseyi in the experimental
facilities of
EWOS Innovation, Puerto Montt, Chile.
Copepodids for the assay were obtained from the rearing unit of Caligus
rogercresseyi in Fundacion Chile experimental facilities, located in Puerto
Montt,
Chile. The sea lice used in this assay were obtained after 9 generations of
Caligus,
reared under control conditions without any exposure to anti-sea lice
compounds.
Twenty eight (28) Petri dishes were filled with 40 ml of microfiltered (5 pm),
aerated
and sterilized seawater (with UV light at 70 pW/cm3). At the initiation of the
assay,
10 copepodids were placed in each Petri dish. Seven doses of Carvacrol were
tested
using four replicates per dose. The doses tested were: 0, 1.5, 3.5, 5.0, 7.5,
10 and
20 pUL. The control group included copepodids only exposed to sea water.
All the Petri dishes were placed into an incubation chamber with constant
temperature set at 14 C. The mortality of the copepodids was evaluated at 0,
1, 6,
16, 24 and 48 hours of incubation. The description of the different treatments
is
shown in Table 1.
=
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Table 1. Concentrations of Carvacrol used to estimate the LD50 of C.
rogercresseyi
in Petri dishes with 40 mL of sterilized sea water (SW).
Concentration Dose
of Carvacrol pUL in 40 mL SW
pUL 40 mL 200 mL*
Control 0 0
1.5 0.06 0.30
3.5 0.14 0.70
5.0 0.20 1.00
7.5 0.30 1.50
10 0.40 2.00
0.80 4.00
* Volume of 200 mL with Carvacrol was prepared to use in each replica
5
The data obtained were analyzed using the R language and its corresponding
packages. LD50 was modelled with the help of a generalized linear model
using binomial model with Iogit link.
Results and Discussion
The data shows that the highest dose of Carvacrol (20 pUL) induced 100% of
mortality after 3 h of exposure (Figure 1). Moreover, it was observed that at
doses
above 7.6 pL/L, the copepodids become lethargic after few minutes of exposure
to
Carvacrol (> 2min).
As a monoterpenoid, Carvacrol is lipophilic and therefore we believe that a
possible
mechanism of action is by passing through the cell wall and cytoplasmatic
membrane, and disrupting structural layers of polysaccharides, fatty acids and
phospholipids of the cells. No negative effect of Carvacrol has been reported
for
salmonids.
It can be observed that there is a time-dose response curve, indicating that
as the
concentration of Carvacrol increased, the mortality of sea lice occur earlier
on time
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(Figure 2). All doses above 5 pL/L induced 100% mortality of the copepodids
(Figure
2).
Table 2. Estimated LD50 values of Carvacrol on copepodids of C. rogercresseyi
for
each exposure time from the categorical time model. Mean, median and 95%
confidence interval (pL/L).
Exposure time (h) Mean Median 95% interval
3 6.44 6.44 5.49 - 7.61
6 5.47 5.49 4.52 - 6.43
16 4.30 4.33 3.61 -4.90
24 3.81 3.84 3.03 - 4.60
48 2.58 2.62 1.87 - 3.33
The results of the LD50 analysis are shown in Table 2. It can be observed that
after
48 h of exposure to Carvacrol the LD50 for copepodids has lower and upper
confidence limits of 1.87 pL/L and 3.33 pL/L, respectively with an estimated
LD50 of
2.58 pL/L.
In the present LD50 assay it was possible to obtain narrow confidence limits,
which
were not far from the estimated LD50 (2.58 pL/L) obtained. This result
increases the
accuracy of the estimate.
Results showed that Carvacrol induced a high mortality of copepodids of
Caligus
rogercresseyi at all exposure time tested in the present experiment (Table 2).
The following conclusions could be obtained from this experiment:
1. Carvacrol induces high mortality over copepodids of Caligus rogercresseyi
at
the concentrations tested in this experiment.
2. The estimated LD50 for Carvacrol was 2.58 pL/L, with narrow confidence
limits.
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Example 2 - Effect of Carvacrol on adult stages of C. rogercressevi
In example 1 it was possible to show that Carvacrol had anti-sea lice activity
against
copepodids stages of Caligus rogercresseyi. Therefore, the next step was to
determine if Carvacrol has a similar activity against adult stages of C.
rogercresseyi.
The objective of this experiment is to determine the LD50 of Carvacrol against
adults
of Caligus rogercresseyi in vitro.
Material and Methods
During January 2009 a study was conducted to determine the LD50 of Carvacrol
against adult stages of Caligus rogercresseyi in the experimental facilities
of EWOS
Innovation, Puerto Montt, Chile.
Adult Caligus for the assay were obtained from the rearing unit of Caligus
rogercresseyi in FundaciOn Chile, experimental facilities, located in Puerto
Montt,
Chile. The adults used in this assay were obtained after 9 generations of
Caligus
reared under control conditions without any exposure to anti-sea lice
compounds.
Twenty four (28) Petri dishes were filled with 40 mL of microfiltered (5 pm),
aerated
and sterilized seawater (with UV light at 70 pW/cm3). At the initiation of the
assay, 10
adults were placed in each Petri dish. Seven doses of Carvacrol were tested
using
four replicates per dose. The doses tested were: 0, 1.5, 3.5, 5.0, 7.5, 10 and
20 pL/L.
The control group included adult Caligus held in Petri dishes with seawater
only.
All the Petri dishes were placed into an incubation chamber with constant
temperature set at 14 C. Sea lice mortality was evaluated at 0, 1, 2, 3, 6,
12, 24, 36
and 48 hours of exposure to the active compound under test. The different
treatments are shown in Table 3.
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Table 3. Concentrations of Carvacrol used to estimate the LD50 of C.
rogercresseyi
in Petri dishes with 40 mL of sterilized sea water (SW) during the assay:
Concentration Dose of Carvacrol
Carvacrol (pL/L)
pL/L 40 mL 200 mL*
Control 0 0
1.5 0.06 0.30
3.5 0.14 0.70
5.0 0.20 1.00
7.5 0.30 1.50
0.40 2.00
0.80 4.00
*Volume of 200 mL with Carvacrol was prepared to use in each replica
5 The data obtained were analyzed using the statistical program SPSS
version 13Ø
The statistical test used to determine the LD50 was PROBIT.
Results and Discussion
Similar to the results obtained in the previous study with copepodids, in the
present
10 experiment, Carvacrol induced a high mortality of the adult stages of
Caligus
rogercresseyi at all concentrations tested (Figure 3).
All the doses of Carvacrol tested in this bioassay killed 100% of the sea lice
at 48 h
of exposure. The highest dose (20 pL/L,) induced 100% of mortality in 2 hours
15 .. (Figure 3). Furthermore, a dose of 10 pL/L induced 100% of mortality
after 12 hours
of exposure (Figure 3). Comparing the results of this bioassay with the
previous
study with copepodids, it shows that the adult stages of C. rogercresseyi
might be
more resistant to Carvacrol than the juveniles.
20 It can be observed that there is a time-dose response, indicating that
as the
concentration of Carvacrol increased, the mortality of adults sea lice occur
earlier on
time (Figure 4).
The results of the LD50 analysis for each exposure time are shown in Table 4.
The
estimates of LD50 doses vary widely and non-systematically between exposure
times (Table 4). It can be observed that after 12 h of exposure, time with a
minor
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variability, the LD50 has a lower and upper confidence limits of 0.45 pL/L and
394.36
pL/L, respectively with an estimated LD50 of 8.26 pL/L.
The following conclusions could be obtained from this experiment:
1. Carvacrol proved to induce high mortality over adults of Caligus
rogercresseyi at the concentrations tested in this experiment.
2. The estimated LD50 obtained after 12 h of exposure was 8.26 pL/L.
Table 4. Estimated LD50 values of Carvacrol on adults of C. rogercresseyi for
each
exposure time from the categorical time model. Mean, median and 95% confidence
interval.
Exposure time (h) Mean Median 95% interval
6 6.00 10.20 0.17 - 294.49
12 8.26 7.97 0.45 - 394.36
24 0.27 5.16 0.01 - 737.58
36 2.43 2.00 0.00 - 25184.62
48 1.46 5.85 0.00 - 1117788.22
Example 3 - Effect of .a fish feed containing Carvacrol on sea lice
According with the results obtained in the examples 1 and 2, Carvacrol has a
surprisingly high potential to reduce sea lice (C. rogercresseyi) infestation.
No
previous studies have been done to test this phenolic compounds, e.g.
Carvacrol
and/or Thymol, directly against sea lice. Further, no previous studies have
been
done to assess the efficacy of Carvacrol and Thymol against sea lice, when
fish are
fed a feed containing such compounds.
The objective of this study is to assess the efficacy of Carvacrol and Thymol
against
sea lice (Caligus rogercresseyi) by feeding fish with a fish feed containing
different
doses of phenol compounds, i.e. Carvacrol and Thymol.
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Material and Methods
The study was conducted in August of 2009, in the facilities of Fundacion
Chile,
Quillaipe, Experimental Station, Puerto Montt, Chile.
5
Four hundred eighty (480) fish (-390 g/fish) were pit-tagged and allocated in
twelve
(12) tanks of 0.35 m3, 40 fish/tank, with sea water filtrated (60 pm). An
acclimatization period of 7 days was performed with a commercial feed. At day
8
commercial feed was changed for the experimental feeds (see table 5 below): a)
10 control, b) 0.17% Carvacrol+Thymol in feed and c) 0.61% Carvacrol+Thymol
in feed.
Four replicas were applied per each treatment. After 12 days of feeding the
fish with
the experimental feeds, salmons were infested with copepodids of C.
rogercresseyi
(with an average infestation rate of 118 copepodids/fish) in 12 tanks of 0.35
m3 with
open sea water flow system and an average temperature of about 13 C. Ten (10)
15 days after infestation, 20 fish per tank were sampled to count the
number of sea lice
attached, where previously fish were anaesthetized with AQUI-S (BAYER,
Germany)
in a bath for about 5 minutes. Sea lice were counted with the fish alive by 3
trained
technicians without the information of the distribution of treatments in the
different
tanks.
Table 5: Basal diet composition showing additionally the inclusion of
Carvacrol and
Thymol ( /0) for the two experimental diets.
Experimental diet
1 2
Dry Matter (%) 94.20 94.20
Crude Protein (%) 44.50 44.50
Lipid (`)/0) 27.30 27.30
Ash (%) 8.10 8.10
Nitrogen Free Extracts (%) 14.30 14.30
Phosphorus (%) 1.30 1.30
Carvacrol and Thymol (%) 0.17 0.61
Results and Discussion
After 10 days of infestation, it was obtained a high reduction in the total
sea lice
attached to the fish, when fish were fed a feed with Carvacrol+Thymol in
comparison
to the control feed (Figure 6).
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Table 6 gives estimates for the percentage reduction in total Caligus counts
for the
Carvacrol+Thymol doses in comparison to the control.
Table 6. Proportional ( /0) reduction in the expected total Caligus counts by
two
different doses of Carvacrol+Thymol in feed in comparison to the control feed
and
the probability of reduction (Pr(x>0)).
Product in feed Dose (/0) Mean reduction CYO Pr(x>0)
Carvacrol+Thymol 0.17 23.7 0.76
Carvacrol+Thymol 0.61 22.6 0.74
For Carvacrol+Thymol, increasing the dose from 0.17 to 0.61 did not appear to
have
any additional effect on Caligus count, being a 23.7% and 22.6% reduction,
respectively (Table 6). The product tested (both doses) had an average
reduction of
about 24% and a moderate probability (81%) support a real effect (Table 7).
Table 7. Proportional (%) reduction in the expected total Caligus counts of
Carvacrol+Thymol in feed in comparison to the control feed and the probability
of
reduction (Pr(x>0)).
Product in feed Mean reduction (%) Pr(x>0)
Carvacrol+Thymol 24.1 0.81
The following conclusions could be obtained from this experiment:
1. Carvacrol+Thymol reduces the infestation of Caligus rogercresseyi, when the
fish were treated with Carvacrol and Thymol added as a component of the
feed composition.
2. A dose of 0.17% of Carvacrol+Thymol in feed, supplied for 22 days to
Atlantic
salmon (390 g/fish in average), reduced sea lice (C. rogercresseyi)
infestation
in about 24%.
From the previous results, it can be concluded that the Carvacrol was
effective in the
control and reduction of parasites that area pathogenic in salmons.
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The experiments clearly show the effect of Carvacrol and/or Thymol directly on
the
fish parasite C. rogercresseyi, and also indirectly when Carvacrol and/or
Thymol
were given to the fish in the fish feed composition. The present invention
thus
demonstrates the surprising potential of these compounds in the treatment and
prevention of parasite infections.
Example 4 - effect of Carvacrol in the reduction of Cafiat's rogercresseyi
infestation
The object of the study was to measure the efficacy of Carvacrol in the
reduction of
Caligus rogercresseyi infestation, when Atlantic salmon (Salmo salar) fed a
feed with
the compound in two different doses.
528 Atlantic salmons were initially allocated in 4 tanks of 3 m3, with 132
fish per tank
and a maximum density of 16 Kg/m3, with flow trough system and filtrated sea
water
.. (60 pm). Fish were pit-tagged and weighted during the allocation in each
tank. During
14 days fish were recovered (7 days) and acclimatized (7 days) in the same
tanks,
feeding a commercial feed. Then, Atlantic salmons were challenged with
copepodids
of C. rogercresseyi (72 copepodids/fish) and 20 fish were sampled (basal
sampling)
to obtain liver, mucus (5 mL), muscle (each fillet) and skin. Samples were
stored and
frozen at -80 C for further chemical analysis. After 10 days of infestation,
all the fish
were sampled to count the number of sea lice per each fish (35 fish/tank).
Then, fish
were allocated in 15 tanks, 0.35 m3/tank, with 35 Atlantic salmon per each
tank. For
20 days, fish were fed three (3) feeds: a) control feed, b) low dose of
Carvacrol in
feed (30 mg Carvacrol/Kg feed) and c) high dose of Carvacrol in feed (120 mg
.. Carvacrol/Kg feed). The source of Carvacrol used in the present experiment
was a
mixture of 50:50 wt/wt Carvacrol+Thymol. The last day (day 20), the level of
infes-
tation was measured by counting the sea lice in all the fish. Fish were
individually
weighted and samples of 5 fish per tank were taken to obtain the liver, mucus
(5mL),
muscle (each fillet) and skin. Samples were stored and frozen at -80 C for
further
chemical analysis of Carvacrol concentration in each tissue.
Mortalities were recorded daily. Environmental variable per each tank were
recorded,
four times per day, specifically dissolved oxygen in sea water (mg/L) and
water
temperature ( C).
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Table 8. Different type of feed tested in the trail and the replicas used per
each
treatment
Diets/ no. of replicates validation efficacy trial (44 1
d_ays):
Control 5
30 mg Carvacrol /Kg feed 5
120 mg Carvacrol /Kg feed 5
No. of units (totally): 15
Statistical analysis
The data obtained were analyzed using the R language and its corresponding
packages. In order to compare lice count between
different test reeds the lice counts made at 3b individual sampled fish were
converted to a total count per tank by summing the individual counts (given
the
number of fish sampled is the same for all tanks). This method is for unbound
counts, such as sea lice counts, and they are assumed to follow Poisson
distribution.
Lice counts can also be overdispersed, i.e. the variance does not follow the
standard
Poisson distribution. This does not affect the coefficient estimates as such
but only
their standard errors. Therefore the model was a generalized linear model
estimated
with the glm function of the R language. The family is Poisson as
overdispersion was
modeled. Treatment estimates were based on posterior simulation (n=2500) with
95% credible intervals as absolute and proportional to the reference level.
Posterior
estimates were averaged over the possible covariate and blocks (http://cran.r-
project.org/doc/manuals/fullrefman.pdf).
Results
During the trial, the water temperature in the tanks varied between 17.8 C and
12.7
C and dissolved oxygen varied from 100% and 80% saturation. Feed consumption
of Atlantic salmon showed a similar trend between the treatments (control, low
dose
and high dose) (Figure 6). The Specific Feeding Rate (SFR) varied between 0.4%
and 0.9% with and average of 0.7%, with the lower consumptions observed in the
first days of the trial and the higher consumption towards the end of the
study.
At the end of the trial the lice infestation In the Atlantic salmon (S. salar)
showed a
high variability between the replicate tanks within each treatment, what is
not
unusual in this type of studies. Counts range of about 2-fold within
replicates (Table
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9). Because of this large variability the estimates of dose effects showed
wide
credible intervals (Table 9) (Figure 7). The results indicate that compared
with the
control group the probability to obtain a better response (reduction of sea
lice
infestation) with the doses of 120 (mg/Kg) and 30 (mg/Kg) of Carvacrol are
close to
70% and 80%, respectively. Not significant statistical differences were
observed
between the treatments (p> .05).
Table 9. Posterior analysis results, when doses (low and high) were compared
with
the control. ("," indicates thousands).
Posterior results: Original scale Proportional to reference Probabitty
of redudng
Diets Mean Median Lower 95% Upper 95% Mean Median Lower95% Upper
95% 1 r(x>1).. lice: 1- Pr(x>1)
0 1,123 1,120 864 1,466 1.02 1.00 0.69 1.44 50% 50%
30 953 953 719 1,269 0.87 0.86 0.57 1.25 21% 79%
120 1,011 1,009 767 1,336 0.92 0.90 0.60 1.32 30% 70%
Example 5 - Efficacy of Carvacrol and Thvmol aoaintt Callous rooercressevi in
rainbow trout (Onchorhynchus mvkiss).
The effect of carvacrol and thymol against the different developmental stages
of
Caligus rogercresseyi has been assessed and the dose of carvacrol and thymol
in
the feed examined to significantly reduce the infestation of sea lice (Caligus
rogercresseyi) in Rainbow Trout (0. mykiss).
Material and Methods
The effect of a synthetic form of carvacrol and thymol over the infestation
rate of
Caligus rogercresseyi in Rainbow trout (0. mykiss) was assessed in two
experiments conducted in sea cages. The first trial (Experiment 1) was
conducted in
a commercial sea site located in the XI Region of Chile. In this experiment,
three (3)
dietary treatments were compared: 1) a commercial control feed (EWOS Silva
1500
and 2500) with no supplementation of any kind; 2) the same base formulation as
in
1, but supplemented with two functional components, nucleotides and a
prebiotic
(EWOS pre-harvest); and 3) the same as in treatment 2, but supplemented with
30
g/ton of feed (0.003%) of pure carvacrol and thymol (50:50 wt) (pre-harvest +
new).
The base formulation of the feeds contained as the major ingredients fish
meal,
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poultry by-products, soybean products, sunflower meal, corn gluten, wheat,
wheat
gluten and a mix of fish and vegetable oils (94.4% of the formulation).
The control, pre-harvest (PH) and pre-harvest + carvacrol and thymol (PH +
new)
5 feeds, were delivered to 7, 3 and 4 cages, respectively. All cages
belonged to the
same site. Each cage contained approximately 50.000 fish. The experiment was
conducted from November 1, 2009 to February 28, 2010. The fish weight
increased
from 1.7 kg to 2.5 kg during the experimental period. During this period, two
pulses
with the dietary treatments were given to the trouts. The first pulse was from
10 November 16 to December 07, 2009 and the second pulse was from January
20 to
February 9, 2010. Caligus counts were recorded on 10 fish per cage once a week
during the two pulses. The measurements of lice infestation were performed in
two
cages per treatment. The life stages of lice recorded in each fish were total
juveniles,
total adults, gravid females and total lice.
The second trail (Experiment 2) was performed in January 2011 in the X Region
of
Chile, at the experimental sea site of EWOS Innovation. The site has 16 cages
of 15
m x 15 m with an automatic feeding system. In this Experiment, Rainbow trout
(O.
mykiss) were used in all cages. The average fish weight at the beginning of
the trial
was 2.0 kg. Each cage contained approximately 10,000 fish. During this trial,
four (4)
dietary treatments were evaluated. One of the feeds was a commercial Control
feed
(EWOS Silva 2000) with no supplementation of any kind. Diets 2, 3 and 4 had
the
same base formulation as the control feed, but supplemented with a mix of
nucleotides, prebiotic, vitamin C and an immune stimulant (EWOS boost). These
three diets were supplemented with 30,45 and 120 9/ton of feed of carvacrol
and
thymol (50:50% wt), respectively. The base formulation of the feeds contained
as the
major ingredients fish meal, poultry by-products, soybean products, sunflower
meal,
corn gluten, wheat, wheat gluten and a mix of fish and vegetable oils (94,6%
of the
formulation).
The source of carvacrol and thymol used was the same as in Experiment 1 and
contained 25% carvacrol and 25% thymol. Therefore, the concentrations of the
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source of carvacrol and thymol in the three experimental feeds were 60, 90 and
240
g/ton of feed, respectively. Thus, as a percentage of the feed, pure carvacrol
and
thymol were supplemented at 0.003,0.006 and 0.012%, respectively.
The EWOS Innovation sea site is located in a production area with natural
infestation
of Caligus. In order to reduce and level off the level of infestation in all
the fish before
starting the experimental period, a bath treatment with deltamethrin (Alphamax
, 0.2
ml/m3 for 30 min) was performed in all fish in a dose tarpaulin system. After
the bath
treatment, an initial sampling of 30 fish per cage was performed to count the
lice
infestation rate. This initial sampling represented the basal infestation rate
just after
the anti-parasite treatment and before starting feeding the experimental
feeds. All
fish were anesthetized with Aqui-S and then weighted, tagged and counted for
sea
lice, as the standard procedure applied by EWOS Innovation. During each
experimental period, 30 fish per cage were sampled on a weekly basis for sea
lice
count. The sea lice life stages recorded included the juvenile stages
copepodids,
Chalimus I, II, II and IV and the adult stages females, gravid females and
males.
Statistical Analyses
Caligus counts are expected to follow the Poisson distribution but to be
overdispersed. Fitting of such multilevel overdispersed Poisson models is best
done
by MCMC (Markov chain Monte Carlo) methods.
In Experiment 1, the counts of different Caligus life stages were modeled as
overdispersed Poisson and estimated with the MCMCgImm package of the R
language (R: A Language and Environment for Statistical Computing, R
Development Core Team, R Foundation for Statistical Computing, Vienna,
Austria,
2008, ISBN 3-900051-07-0. Diffuse flat normal priors
centered around zero but with a large variance (108) were used for the fixed
effects
and weakly informative inverse Wishart priors for the variance parameters
(random
effects). Similarly, a multilevel model for the average counts over the whole
monitoring period was estimated by using both cage and sampling date as random
covariates. Model was estimated with the MCMCgimm as described above.
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Experiment 2 is a dose-response experiment and the potentially nonlinear
effect of
dose was modeled with a second degree polynomial. Note: day of sampling was
still
considered as a factor, which in practice means the polynomial dose-response
models between different samplings are independent The fitted model was used
to
compute the evolvement of the expected lice counts and the expected
reinfestation
counts (by subtracting the posterior distribution at day = 0). The latter is
Important
since the initial lice counts can vary between the treatments due to the bath
treatment efficacy. The counts of different life stages showed typically
higher
variation between the treatments than the total count. All statistical
modelling was
conducted with the R language and its corresponding packages (R: A Language
and
Environment for Statistical Computing, R Development Core Team, R Foundation
for
Statistical Computing, Vienna, Austria, 2008, ISBN 3-900051-07-0).
Results
Exoeriment 1
Expected average counts over the whole monitoring period for each treatment by
lice
life stage are shown in Figure 8. Differences in the expected average counts
exist
between the treatments. There is a general trend for all life stages that the
PH
treatment has somewhat lower expected count than the control, and the PH+new
treatment (with carvacrol and thymol) had an even lower expected count. For
gravid
females and total sea lice it can be claimed there is strong evidence of lower
counts
for the PH+new treatment (with carvacrol and thymol) in comparison to the
control.
This is especially relevant and interesting since obviously the gravid females
are an
important triggering factor in sea lice population development. A comparable
effect of
lower counts are also observed for the PH treatment when considering gravid
females only. There is a difference between the PH and PH+new treatment in
that
the PH+new diet (with carvacrol and thymol) seems to affect also juveniles and
adults while the PH diet only affects gravid females.
=
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Experiment 2
At the beginning of the trial, the infestation of sea lice (Caligus
rogercresseyi) was
different between the different experimental groups (Table 10).
Table 10. Initial total lice counts per fish for the different treatments in
Experiment 2:
Control Experimental groups
Doses carvacrol + thymol added in feed (ppm)
30 45 120
Counts of sea lice 10.01 12.61 11.27 15.21
(N )
The reinfestation was calculated from the posterior distributions of the model
by
subtracting the posterior distributions of control and experimental treatments
at
day=0 from the distributions of the other days. The reinfestation counts can
be
negative if the lice count of a specific life stage decreased from the initial
count. The
results are shown in Figures 9 and 10. These graphs show that the fish fed
with
carvacrol and thymol had a lower reinfestation rate for juveniles, adults and
total lice
in a dose dependent trend (Figure 9). An inclusion of as low as 0.003% of
carvacrol
and thymol in the feed was surprisingly effective on the reinfestation of sea
lice in
spite of the comparable low dose.
Although the higher dose of carvacrol and thymol had a stronger effect than
the
lower doses tested. A similar effect is observed for gravid females, females
and
males (Figure 10).
To better understand and see the effects of the dose of carvacrol and thymol
on the
different life stages of lice, the linear and quadratic coefficients of the
dose-response
model of each sampling date are plotted together with their 95% credible
intervals
(Figure 11). This Figure deserves some explanation. The linear and quadratic
coefficients describe the shape of the dose effect and the credible intervals
show
how certain such effects are. Therefore, we look for statistically significant
coefficients that have 95% credible interval that does not cross the zero line
marked
to the graph.
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On day 7, such linear coefficients can be seen for Ch 1&II, gravid females,
females,
males and total number of lice, and all are negative, meaning that the
carvacrol and
thymol linearly and significantly reduced the reinfestation of these life
stages. The
linear coefficient implies the dose has a linear effect on the lice counts.
The only
quadratic coefficient that is significant is the positive coefficient for
gravid females but
many other life stages having significant linear coefficient tend to have a
positive
quadratic coefficient. By putting a negative linear and positive quadratic
coefficient
together means that the dose has initially a decreasing effect on lice count
but this
effect levels off at higher doses. On day 14, the picture is very similar to
day 7. More
quadratic coefficients can now be considered significant, i.e. they confirm
the type of
the dose effect explained above for day 7. A similar effect on copepodids is
now
evident as well. There seems now a positive linear coefficient with negative
quadratic
coefficient for Ch 1118,IV indicating the initial increase in the dose has
small or no
effect and only the highest doses may be effective for this life stage. On day
21, the
only significant effects are the negative linear and positive quadratic
coefficients for
gravid females and the opposite for Ch 1118,1V.
Conclusions
The following conclusions can be obtained from this study:
- The dietary supplementation of carvacrol and thymol significantly reduced
the
reinfestation of different life stages of Caligus rogercresseyi during a
period of
3 to 4 weeks in rainbow trouts.
- At very low and higher doses of carvacrol and thymol (0.003 to 0.012%),
the
effect was more evident on the gravid females, while a dose of 0.012% was
required to also reduce the infestation of juvenile stages.
Thus, by way of the present invention it was further demonstrated for the
first time
that there are clearly differences in the efficacy of thymol and carvacrol on
different
life stages of the ectoparasitic copepod sea lice, dependent both on the
period of
application and concentration of the active compounds. Already comparable low
doses are efficient on gravid females while higher doses seem to be necessary
to
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combat juveniles. This is also of practical relevance for the use of the
active
compounds in the treatment of sea lice infections. It suggests that Carvarcrol
and
thymol can be used in lice control where gravid females are specifically
targeted at
or combated in specific periods by using low concentrations of the active
5 compounds.
The efficacy of carvacrol and thymol in the treatment of sea lice is increased
by the
presence of immune stimulants in the feed as shown in example 5. The addition
of
immune stimulants are not restricted to non-specific immune stimulants, but
can also
be selected from specific immune stimulants.
10 It is preferred that the treatment with a feed comprising carvacrol
and/or thymol is
carried out on a continuous basis over an extended period of time, wherein the
active compounds are preferably added to the feed consumed by the fish on a
daily
basis. It is preferred that fish are fed for at least 7 days with the diet,
more preferred
for 3 to 4 weeks. However, longer periods extending 28 days may also be
applied.
It is assumed that the same effects as shown for sea lice will also be
relevant for
other ectoparasitic copepods in fish and that the two active ingredients can
be used
for the treatment and prevention of diseases and infections caused by other
parasitic
copepod infections.
It will be appreciated that the embodiments of the invention described in the
foregoing can be modified without departing from the scope of the invention.
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Definitions used in the application
Carvacrol is the compound 5-isopropyl-2-methylphenol (C6H3CH3(OH)(C3H7)), and
is
a monoterpenoid phenol. It has a characteristic pungent, warm odor of oregano
and
.. a pizza-like taste. It is present in the essential oil of Origanum vulgare,
oil of thyme,
oil obtained from pepperwort, and wild bergamot.
Thymol is the compound 5-isopropyl-2-methylphenol, and is a monoterpenoid
phenol
derivative of cymene, isomeric with Carvacrol, found in oil of thyme, and
extracted as
.. a white crystalline substance of a pleasant aromatic odor and strong
antiseptic
properties.
Prebiotics are food ingredients that stimulate the growth and/or activity of
bacteria in
the digestive system being beneficial to health.
Immune stimulants are substances (drugs and nutrients) that stimulate the
immune
system by inducing activation or increasing activity of any of its components.
They
may be non-specific or specific immune stimulants.
Conventional feed ingredients are feed ingredients which are commonly used in
feed
compositions for a specific animal species such as lipids, proteins, vitamins,
carbohydrates, minerals, etc
Nucleotides comprise any known phosphor ester of a nucleoside such as AMP,
GMP, UMP, CMP, UMP.
