Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHOD AND COMPOSITION FOR CONTROLLING LICE
The present invention relates to a method and device
for controlling lice, in particular sea lice.
Sea louse is the general name of parasitic oar--
footed crustaceans (copepods), which are found at
(marine) water culture production locations where fish
are farmed. In Northern Europe and particularly in
Scotland and Norway sea lice are already the most
significant threat to production locations of sea fish
and the problem only continues to grow. Damage and death
of fish due to sea louse infections are an important cost
overhead in aquaculture. The problem also occurs in
seawater and brackish water fish farms and shrimp ponds
in South-East Asia, particularly in Thailand, the
Philippines and Indonesia.
The parasitic sea louse copepods belong to the
family of the Caliqidae, comprising 23 genera and 200
species. The most important genera are Lepeophteirus,
Caliaus and Pseudocaliqus, because they can result in
high mortality. In Northern Europe the most important
parasite is Lepeo~htheirus salmonis and to a lesser
degree Caliq_us elonaatus. Both belong to the Caliaidae
and are ectoparasites on salmonoids. The life cycle of
L.salmonis comprises 10 stages, of which the 4 chalimus
stages infect the salmon. They attach themselves to the
fish with claw-shaped antennae, penetrate the skin of the
fish and thus cause skin lesions and large open wounds.
Secondary bacterial and fungal infections are
subsequently often the cause of death of the fish.
In order to control the sea lice use is often made
of hydrogen peroxide, which is added to a treatment bath
in a quantity of about 1500 mg/1 (see for instance US-
5,313,911 in the name of Eka Nobel AB). However, both the
large volumes of hydrogen peroxide and the limited
activity and toxicity for the fish do not make this an
ideal method.
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Bath treatments are further also applied with
different types of pesticide such as CypermethrinTM,
NuvanT"' (active substance dichlorvos) , PyrethrumT''' and
Dipterex'~'"' (active substance trichlorophon) . These
substances can only be used under heavy restrictions and
have great drawbacks. They are not only toxic for the
lice, but also harmful to the fish and the environment.
Residues of the substances moreover accumulate in the
fish and thus form an indirect risk to the consumer.
Handling of the substances also involves risks. Finally,
these substances are not active against every stage of
development of the sea lice.
In the light of the above, it is the object of the
present invention to provide an effective, natural and
environmentally-friendly system with which the lice can
be controlled without too many drawbacks for the fish.
This is achieved by the invention with a method for
controlling lice on an organism, comprising of treating
the organism with a composition which at least consists
of
a) lactoperoxidase;
b) thiocyanate; and/or
c) iodide; and
d) a hydrogen peroxide source.
Although this method is particularly suitable for
controlling sea lice, it can also be employed to control
other lice, such as aphids on plants, lice on animals,
such as head lice in people.
The methods of administration in controlling other
types of lice are of course not the same as in
controlling sea lice. In the latter case the agent is
added to water in which the fish are accommodated for a
longer or shorter time or in which they are immersed.
Controlling lice on plants can for instance be done by
spraying, while treatment of head louse can take place by
rinsing, rubbing-in or spraying.
The composition consisting of lactoperoxidase,
thiocyanate and/or iodide and hydrogen peroxide is most
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effective when the concentration thereof with which the
lice come into contact amounts for lactoperoxidase (LP)
to 0.5 to 20 mg/l, preferably 1 to 10 mg/l, more
preferably 2.5 to 7.5 mg/1 and most preferably about 5
mg/l, for hydrogen peroxide to at least 10, preferably at
least 50, more preferably at least 100 mg/1, for
thiocyanate (SCN-) to at least about 2.5 mg/l, preferably
at least about 5 mg/l, more preferably at least about 10_
mg/l, but a maximum of 100 mg/l, and for iodide (I-) to at
least about 5 mg/l, preferably at least about 30 mg/l,
but a maximum of 100 mg/l.
When reference is made in this application to
"concentration with which the lice come into contact",
this is intended to mean the concentration which is
present in the treatment bath in the case of sea lice, or
in the spray or other means of application in the case of
other lice. "Composition" is likewise understood to mean
that in which the concentrations are equal to the
treatment concentrations.
Such a composition for controlling lice on an
organism therefore comprises the following active
substances:
a) lactoperoxidase (LP) in a quantity of 0.5 to 20
mg/l, preferably 1 to 10 mg/1, more preferably 2.5 to 7.5
mg/1 and most preferably about 5 mg/l;
b) hydrogen peroxide in a quantity of at least 10,
preferably at least 50, more preferably at least 100
mg/l;
c) thiocyanate (SCN-) in a quantity of at least about
2.5 mg/1, preferably at least about 5 mg/l, more
preferably at least about 10 mg/l, but a maximum of 100
mg/l; and
d) iodide (I-) in a quantity of at least about 5
mg/1, preferably at least about 20 mg/l, but a maximum of
100 mg/1. In a preferred embodiment the composition
according to the invention comprises: 5 mg/1
lactoperoxidase, 10 mg/1 thiocyanate, 30 mg/1 iodide and
100 mg/1 hydrogen peroxide.
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This composition can in turn be manufactured from a
kit consisting of at least two components.
The components of the kit are at least two
components, wherein the first component comprises
lactoperoxidase, thiocyanate and/or iodide and the second
component hydrogen peroxide. Supplying hydrogen peroxide
separately prevents lactoperoxidase already becoming
active in the packaging. The activity of the final
composition could thereby be reduced. A kit may however
also consist of more than two components, wherein in
addition to the separate hydrogen peroxide the other
constituents are also supplied separately or in pairs of
two. The components can each individually be in liquid or
solid form. Particularly the component consisting of
lactoperoxidase, thiocyanate and/or iodide also forms
part of the invention.
A particularly advantageous embodiment of the kit
comprises at least two components, wherein the first
component is formed by a composition comprising
lactoperoxidase in a quantity resulting in a
concentration with which the lice come into contact of
0.5 to 20 mg/1, preferably 1 to 10 mg/l, more preferably
2.5 to 7.5 mg/1 and most preferably about 5 mg/l,
thiocyanate in a quantity resulting in a concentration
with which the lice come into contact of at least about
2.5 mg/1, preferably at least about 5 mg/l, more
preferably at least about 10 mg/l, but a maximum of 100
mg/1, and/or iodide in a quantity resulting in a
concentration with which the lice come into contact of at
least about 5 mg/l, preferably at least about 20 mg/1,
but a maximum of 100 mg/1, and the second component is
formed by a solution of hydrogen peroxide in a quantity
resulting in a concentration with which the lice come
into contact of at least 10, preferably at least 50, more
preferably at least 100 mg/l. Particularly recommended is
a kit wherein the first component consists of
lactoperoxidase in a quantity resulting in a
concentration with which the lice come into contact of
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about 5 mg/1, thiocyanate in a quantity resulting in a
concentration with which the lice come into contact of
about 10 mg/1 and/or iodide in a quantity resulting in a
concentration with which the lice come into contact of
5 about 30 mg/l, and the second component is formed by a
solution of hydrogen peroxide in a quantity resulting in
a concentration with which the lice come into contact of
100 mg/1.
The composition can also be formed from a
concentrated composition or a solid composition which
contains all constituents. The desired treatment
concentrations then result by adding this concentrated
liquid or solid composition to water.
It is recommended to prepare the treatment bath
prior to the fish being placed therein. This prevents
damage to the fish occurring due to locally high
concentrations of the different constituents which have
not yet dissolved or are not yet well distributed through
the water.
In order to safeguard the health of the fish as much
as possible, the treatment time is preferably kept as
short as possible. Treatment times between 5 and 60
minutes are recommended. Although the agent according to
the invention is much less toxic than the high
concentrations of hydrogen peroxide or pesticides used
heretofore, it is nevertheless recommended to limit the
contact between fish and active substances as much as
possible. The treatment may optionally be repeated at a
later stage in the case control is not complete.
The invention further relates to the use of the
composition, kit or components thereof for controlling
lice in general and sea lice in particular. The invention
also comprises the use of the kit or the components
therefrom for manufacturing the composition.
The invention will be further illustrated with
reference to the examples following below. The
composition according to the invention is herein
designated as "LP system".
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EXAMPLES
EXAMPLE 1
In vitro tests with lactoperoxidase (LP)systems against
sea louse
Egg sacs of the sea louse Lepeophteirus salmonis
were collected and incubated for 5 days at 15°C in water
with a salinity of 3.4o per weight. During this time the_
oar-footed crustaceans developed into healthy specimens.
One or more of the components lactoperoxidase,
iodide, thiocyanate and hydrogen peroxide were dissolved
in seawater sterilized using an ozone treatment and
filtration. About 200 copepodids per litre were
subsequently added to each of these solutions and
incubated for 20 minutes. The copepodids were then
filtered off, washed and placed once again in clean
seawater. The percentage of surviving copepodids was
determined after 1 hour.
The composition of the solutions and the survival of
the copepodids therein are shown in tables 1 and 2.
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Table 1 Effect of individual components
Blank I~Only SCN HZOZ LP OnlyLP Only
Only Only
Lactoperoxidase,0 0 0 0 10 20
mg/1
K-iodide, mg/1 0 30 0 0 0 0
K-thiocyanate, 0 0 10 0 0 0
mg/1
HZOZ, mg/1 0 0 0 200 0 0
% survival after95 88 86 93 90 69
1 hour
Table 2 Effect of LP systems
Lactoperoxidase,0 2.5 5 10 10 10 20 20
mg/1
K-iodide, mg/1 0 30 30 30 30 30 30 30
K-thiocyanate, 0 10 10 10 10 10 10 10
mg/1
1 HyOZ, mg/1 0 100 100 10 50 100 100 200
5
% survival afterg5 73 39 31 6 0 0 0
1 hour
From tables 1 and 2 can be seen that the individual
components have hardly any effect on the sea lice, but
that the combination thereof in the LP system according
to the invention does so.
EXAMPLE 2
Sensitivity of fish to LP systems
Young salmon with an average weight of about 50 g.
were exposed to solutions (in seawater) of the individual
components and to complete LP systems. The table below
shows the concentration.
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components control 1 2 3 4 5 6
group
Lactoperoxidase0 10 0 0 5 2.5 1
mg/1
K-thiocyanate 0 0 10 0 5 2.5 2.5
mg/1
K-iodide mg/1 0 0 0 30 5 7.5 7.5
HZOZ, mg/1 0 0 0 0 100 100 100
The transfer of young salmon to a new environment will in
any case cause stress phenomena, such as a slightly
increased gill cover activity and agitated swimming on
the surface. Calm is virtually restored after about 30
minutes.
These phenomena were observed in both the control
and solutions of the individual components, wherein there
was hardly any difference, or none at all, between the
control and individual components.
The stress reactions with complete LP systems were
clearly higher, at the lowest concentration (1 mg/1 LP)
agitated swimming behaviour and a moderately increased
gill cover activity was still present after 30 minutes.
Only after 60.minutes was calm restored.
At a concentration of 2.5 mg/1 LP these phenomena
were more pronounced but still acceptable.
At a concentration of 5 ppm LP stress phenomena were
even more severe. After 60 minutes the majority of the
fish was still lethargic and swimming in uncoordinated
manner. There were no fatalities however.
A test with larger salmon (about 500 g) and a system
with 5 mg/1 LP showed that these fish were hardly
affected by the treatment.
EXAMPLE 3
In vivo study of the effect of LP systems on fish which
"infected" with sea lice
In this example the effect of an LP system with 2.5
mg/1 LP, 2.5 mg/1 KSCN, 7.5 mg/1 KI and 100 mg/1 H202 is
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studied in a situation in which young Atlantic salmon
were "infected" with sea lice of L. salmonis.
1. Method
In four tanks of lm3 each, 40 young salmon of about
50g in their second year of life were kept per tank in
seawater treated with ozone (>750 Mv) and filtered by
carbon at ambient temperature (14°C ~ 1°C). Per tank the_
fish were brought into contact with 1000 copepodids of L.
salmonis for 1.5 hours and the lice were allowed to
develop to pre-adult stages.
Counts were carried out prior to the treatment and
it was determined that all tanks contained sufficient
pre-adult stage lice. The treatments were then started.
Two of the tanks (1 and 3) were treated with the LP
system according to the invention (2.5 mg/1 LP, 2.5 mg/1
KSCN, 7.5 mg/1 KI and 100 mg/1 H202) for 20 minutes. Tanks
2 and 4 received an identical treatment with seawater.
The temperature of the seawater was 15°C and it contained
more than 8 mg/1 oxygen. Samples were assessed l, 24 and
48 hours after treatment. Lice levels on the fish were
recorded and compared with the levels before the
treatment making use of Student's t-test.
2. Results
The results of the lice counts are shown in the
table below. The lice counts are expressed per fish and
are average values of 10 fish.
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Table 9
Sea lice counts: average values per fish (n=10)
betore treatmentafter treatment,after treatment,after treatment,
1 hour 24 48
hours hours
Tank 4.0 3.3 2. 1.
1
(LP-s) (SEM = 0.494)(SEM = 0.60) (SEM = 0.64) (SEM = 0.5)
an 2 4.8 4.4 4.2 .4
(control)(SEM = 0.629)(SEM = 0.56) (SEM = 0.61) (SEM = 0.4)
Tank 4.7 4.0 2.1 2.2
3
(LP-s) (SEM = 0.731)(SEM = 0.56) (SEM = 0.41 ) (SEM = 0.2)
Ta 4 3.5 5.1 3.7 3.8
(control)(SEM = 0.401)(SEM = 0.74) (SEM = 0.94) (SEM = 1.08)
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Lice levels
1 hour after the treatment there was no significant
reduction in lice levels in any of the groups. After 24
hours both treated groups had significantly fewer lice.
48 hours after the treatment there was a reduction of
respectively 63% and 53% (p<0.01). There was no
significant reduction in lice levels in untreated control
groups.
Behaviour of the fish
In the eleventh minute during the treatment the fish
displayed some agitation with an increase to rapid
swimming and jumping activity at 15 minutes. At 19
minutes some fish were at the point of death and only
recovered after the tank had been flushed. There were no
fatalities.
The treatment with an LP system consisting of 2.5
mg/1 LP, 2.5 mg/1 KSCN, 7.5 mg/1 KI and 100 mg/1 H202 for
minutes was on average 58o effective against the sea
lice. There appeared to be some effect on the fish, but
this was not fatal. Because small fish were treated here
20 at a high seawater temperature, this test was performed
under the worst possible conditions. Larger fish at a
lower temperature will be more resistant to the effects
of an LP system.
