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 Salmo 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 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. 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 rogercresseyi.

Preferable, the ratio of carvacrohthymol 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 Artie 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. 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 rogercresseyi. 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 Artie charr (Salvelinus alpinus).

Preferable, said active compound, mixture of compounds or composition is applied to a locus to be protected from the parasites. 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 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 carvacrohthymol 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 Artie charr (Salvelinus alpinus). 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. 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 % 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.

Detailed description of the invention

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 (pL/L) 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. Figure 5 shows the proportion (%) 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. Experimental section

Example 1 - Effect of Carvacrol on copepodids of Caligus rogercresseyi

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 μιη), aerated and sterilized seawater (with UV light at 70 W/cm ³ ). 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 μΙ_/Ι_. 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 . Table 1. Concentrations of Carvacrol used to estimate the LD50 of C. rogercresseyi in Petri dishes with 40 ml_ of sterilized sea water (SW).

* Volume of 200 mL with Carvacrol was prepared to use in each replica

The data obtained were analyzed using the R language and its corresponding packages (http://www.r-project.org/). LD50 was modelled with the help of a generalized linear model using binomial model with logit link (http://cran.r- project.org/doc/manuals/fullrefman.pdf).

Results and Discussion

The data shows that the highest dose of Carvacrol (20 pL/L) induced 100% of mortality after 3 h of exposure (Figure 1 ). Moreover, it was observed that at doses above 7.5 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 (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%

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. Example 2 - Effect of Carvacrol on adult stages of C. rogercresseyi

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/cm ³ ). 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.

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:

* Volume of 200 mL with Carvacrol was prepared to use in each replica The data obtained were analyzed using the statistical program SPSS version 13.0. 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 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 μί/L,) induced 100% of mortality in 2 hours (Figure 3). Furthermore, a dose of 10 μί/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. 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 variability, the LD50 has a lower and upper confidence limits of 0.45 μΙ_/Ι_ 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.

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. Material and Methods

The study was conducted in August of 2009, in the facilities of Fundacion Chile, Quillaipe, Experimental Station, Puerto Montt, Chile.

Four hundred eighty (480) fish (~390 g/fish) were pit-tagged and allocated in twelve (12) tanks of 0.35 m ³ , 40 fish/tank, with sea water filtrated (60 μπι). 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) 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 m ³ with open sea water flow system and an average temperature of about 13°C. Ten (10) days after infestation, 20 fish per tank were sampled to count the number of sea (ice 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 (%) for the two experimental diets.

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). 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 (%) 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)).

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)).

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. 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 Caliaus 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 m ³ , with 132 fish per tank and a maximum density of 16 Kg/m ³ , 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 m ³ /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 infestation 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). Table 8. Different type of feed tested in the trail and the replicas used per each treatment

Statistical analysis

The data obtained were analyzed using the R language and its corresponding packages (http://www.r-project.org/ ). In order to compare lice count between different test feeds the lice counts made of 35 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 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).

Example 5 - Efficacy of Carvacrol and Thymol against Caligus rogercresseyi 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 (O. mykiss).

Material and Methods

The effect of a synthetic form of carvacrol and thymol over the infestation rate of Caligus rogercresseyi in Rainbow trout (O. 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, 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) 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

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 20 1 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 g/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 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/m ³ for 30 min) was performed in all fish in a close 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 MCMCglmm 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, http://www.R-project.org). 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 MCMCglmm as described above. 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, http://www.R- project.org). Results

Experiment 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. 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:

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 1 1 ). 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. On day 7, such linear coefficients can be seen for Ch l&ll, 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 lll&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 lll&IV.

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 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

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.

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.

Definitions used in the application

Carvacrol is the compound 5-isopropyl-2-methylphenol (C6H ₃ CH3(OH)(C ₃ H ₇ )), 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.