NO20121254A1 - Compound and composition for controlling parasites - Google Patents

Abstract

Carvacrol and / or thymol or a composition comprising one or both of these active compounds are disclosed for use in the treatment of fish lice attacks and a method for killing, fighting or regulating lice, preferably in the fish farming industry, and also a feed for fish.

Description

The field of the invention

The present invention relates to carvacrol and/or thymol, or a composition comprising one or both of the active compounds, and a method for killing, combating or controlling lice in fish. The present invention further relates to the use of carvacrol and/or thymol for the production of a pharmaceutical composition for feeding a fish.

Background for the invention

Infectious parasitic disease outbreaks are a serious problem in aquaculture of many different species. Economic losses caused by these diseases can be severe and considerable research efforts have been dedicated to treating and preventing these diseases in aquaculture production. In addition, the parasitic infestations in farmed aquatic animals are not only harmful to the farmed animals, but they also represent a constant and serious threat to wild populations that can be infected by farmed animals. This is another reason why the 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 marine lice ("sea lice") have for many years and until today constituted a growing problem in salmon production in countries such as Norway, Chile, Scotland, Ireland and Canada. Attacks on farmed fish, especially Atlantic salmon, Salmo salar, by the ectoparasitic copepods Lepeophtheirus salmonis, Caligus elongatus and Caligus rogercresseyi are not only harmful to the fish, but they can eventually also lead to increased mortality in severe cases of infection.

Attacks by lice (for example Caligus rogercresseyi and L. salmonis) are currently one of the most important health challenges for salmon production. This external parasite impairs production efficiency, causes stress and damage to the fish and, as a secondary infection, is a possible vector for other pathogens, such as salmon alphavirus (for Pancrease Disease), infectious salmon anemia virus (ISAv) and Piscirickettsia salmonis. In Chile, as in many other salmonid producing countries, the frequency of attacks has increased significantly and mainly affects species such as Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and silver salmon (Oncorhynchus kisutch). In Norway, lice infections are considered one of the most serious health problems in salmon aquaculture. This higher attack rate is probably a consequence of a combination of factors, including the fact that the louse has developed widespread resistance to most of the chemical antiparasitic agents used in the treatment as of today.

Commonly used chemotherapeutic agents for the treatment of lice infections belong, for example, to the group of organophosphates, pyrethroids, hydrogen peroxides, chitin synthesis inhibitors and emamectin. However, resistances, which also often include multiple and cross-resistances, often result in limited success when using many of the above-mentioned commonly used anti-lice compounds.

The toxicity of many pharmacologically effective compounds to the treated animals, as well as a limited biodegradation, is limiting for their use for aquaculture purposes. Preferably, the compounds should be non-toxic to the fish in the concentrations used, and due to environmental issues, the compounds should be rapidly and easily degradable in the natural environment without any undesirable and harmful accumulation in the food chain. When supplied through the diet, the compounds should either be broken down or excreted by the organism after a reasonable period of time. This is particularly important because farmed animals are ultimately consumed by humans.

Therefore, it is very important to find new compounds and ways to treat and prevent ectoparasite infections in fish, especially lice.

The objective technical problem of the present invention is to provide new effective means and procedures for killing copepod ectoparasites, and/or inhibition of attacks by ectoparasites in fish, such as 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 these active compounds for use in the treatment and/or prophylaxis of ectoparasitic copepod infection and/or attack in a fish.

Preferably, said ectoparasitic copepod is a louse.

Preferably, said louse is selected from Lepeophtheirus salmonis, Caligus elongatus and Caligus rogercresseyi.

Preferably, the carvacrothymol ratio is in the range of 1:3 to 3:1, more preferably 1:2 to 2:1, most preferably approx. 1:1 based on weight.

Preferably, said fish is a salmon fish, and more preferably a fish selected from the group consisting of Atlantic salmon {Salmo salar), rainbow trout {Onchorhynkuss mykiss), Coho salmon (Oncorhynchus kisutch) and arctic char (Salvelinus alpinus).

Preferably, the active compounds are used for reduction in the total number of adult forms of lice that attack or re-infest the fish.

Preferably, carvacrol and/or thymol are added to a preformulation in a total concentration of between 0.001% to 0.8% of the feed, preferably 0.003% to 0.6% and more preferably between 0.003 and 0.012% by weight of the feed.

Preferably, the total concentration of carvacrol and/or thymol is 30 mg/kg or more, more preferably 45 mg/kg for or more, more preferably at least 100 mg/kg and most preferably at least 120 mg/kg for.

Preferably, carvacrol and thymol are 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 more of the following ingredients selected from the group consisting of nucleotides, prebiotics, vitamin C and immunostimulants, which may be specific or non-specific.

Preferably, the compound or composition is supplied to fish for at least 7 days, more preferably at least 21 days and most preferably at least 28 days.

Preferably, the treatment with the active compounds is combined with other chemical and medicinal agents against lice.

Preferably, the active compounds are used specifically to combat certain life forms of the louse.

Preferably, the active compounds are used to reduce the attack of pregnant female bedbugs on the fish.

Preferably, the fish are fed continuously with the feed comprising the active compounds as part of their daily diet.

Preferably, the active compound or compounds are applied in the form of a water bath treatment.

Another aspect of the present invention relates to a method for killing, controlling or controlling ectoparasitic copepods, the method comprising applying carvacrol and/or thymol or a composition comprising one or both of the active compounds to kill the lice, to make them drowsy or to prevent attacks on the fish.

Preferably, said ectoparasitic copepod is a louse.

The lice Lepeophtheirus salmonis, Caligus elongatus or Caligus rogercresseyi are preferred.

Preferably, said fish is a salmon fish, preferably selected from the group consisting of Atlantic salmon {Salmo salar), rainbow trout {Onchorhynkuss mykiss), Coho salmon (Oncorhynchus kisutch) and arctic char (Salvelinus alpinus).

Preferably, said active compound, mixture of compounds or composition is applied to a locus to be protected against the parasites.

Preferably, said locus is an aqueous environment where the active compound or composition comprising the active compound is applied directly to the aqueous locus.

Preferably, said locus contains fish and where said lice are located on said fish or said fish is susceptible to attack or infection by said lice, where the active compound or mixture of compounds is applied directly to the fish, preferably by topical or oral application.

Preferably, the active compound or mixture of compounds is applied to the fish as a component of the feed given to the fish.

Preferably, the carvacrothymol ratio is in the range of 1:3 to 3:1, more preferably 1:2 to 2:1, and most preferably about 1:1, based on weight.

Preferably, one or more of the active compounds or a composition comprising the active compounds is supplied to the fish as part of the feed for at least 7 days, more preferably at least 21 days, and most preferably at least 28 days.

A third aspect of the present invention relates to the use of carvacrol and/or thymol for the preparation of a pharmaceutical composition for the treatment and/or prophylaxis of ectoparasitic copepod infection and/or attack in a fish.

Preferably, said ectoparasitic copepod is a louse.

Preferably, said louse is selected from Lepeophtheirus salmonis, Caligus elongatus and Caligus rogercresseyi.

Preferably, the carvacrothymol ratio is in the range of 1:3 to 3:1, more preferably 1:2 to 2:1, most preferably approx. 1:1 based on weight.

Preferably, said fish is a salmon fish.

More preferably, said fish is selected from the group consisting of Atlantic salmon (Salmo salar), rainbow trout (Onchorhynkuss mykiss), Coho salmon (Oncorhynchus kisutch) and arctic char (Salvelinus alpinus).

Preferably, the pharmaceutical composition is for reducing the total number of adult lice forms that attack or re-infest the fish.

Preferably, the pharmaceutical composition comprising carvacrol and/or thymol is added to a preformulation 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 preferably from 0.003 and 0.012% by weight of the feed.

Preferably, the pharmaceutical composition comprising carvacrol and/or thymol is added to a pre-composition, which has a total concentration of carvacrol and/or thymol of at least 30 mg/kg for, preferably at least 45 mg/kg for, more preferably at least 100 mg/kg and most preferably at least 120 mg/kg for.

Preferably, said carvacrol and thymol are in a 1:1 ratio.

Preferably, the feed comprises other conventional feed ingredients in the fish feed.

Preferably, the feed further comprises one or more of the following ingredients selected from the group of nucleotides, prebiotics, vitamin C and immunostimulants, which may be specific or non-specific.

Preferably, the compound or composition is supplied to the fish for at least 7 days, more preferably at least 21 days and most preferably at least 28 days.

Preferably, the treatment with the active compound is combined with other chemical or medicinal agents against lice.

Preferably, the active compounds are used specifically to combat certain life forms of the louse.

Preferably, the active compounds are used to reduce the attack of pregnant female bedbugs on the fish.

Preferably, the fish are continuously fed feed containing the active compounds as part of their daily intake.

Preferably, the pharmaceutical composition is applied in the 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 preferably between 0.003 and 0.012% by weight of lined.

Preferably, the total concentration of carvacrol and/or thymol is at least 30 mg/kg, preferably at least 45 mg/kg for, more preferably at least 100 mg/kg and most preferably at least 120 mg/kg for.

Preferably, the carvacrothymol ratio is in the range of 1:3 to 3:1, more preferably 1:2 to 2:1, most preferably approx. 1:1 based on weight.

Detailed description of the invention

Embodiments of the invention will now be described using examples where reference is made to the following figures, in which;

Figure 1 shows the observed mortality of copepodites of C. rogercresseyi (in relation to the number of lice at the start of the study), over a period of 48 hours 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 copepodites of C. rogercresseyi exposed to different doses of carvacrol (ul/l) during 48 hours. "Error bars" represent standard deviations obtained for the replicates at each dose of carvacrol. Figure 3 shows the observed mortality of adult C. rogercresseyi (1 relative to the start) over a period of 48 hours at different doses of carvacrol. Figure 4 shows the percentage mortality of C. rogercresseyi adults exposed to different doses of carvacrol (pl/1) during 48 hours. "Error bars" represent standard deviations obtained for the replicates at each dose of carvacrol. Figure 5 shows the ratio (%) for reduction in expected lice (C. rogercresseyi) attacks on the fish when they are fed two doses of carvacrol + thymol () in the feed compared to the control feed. Figure 6 shows (for example 4) the feed consumed by the fish in each treatment throughout the experiment, a) Specific feeding rate (SFR) for fish fed with a high dose of carvacrol, b) SFR for fish fed with a low dose of carvacrol and c) SFR for fish fed with control feed. Figure 7 shows (for example 4) lice (C. rogercresseyi) infestations expressed as total number of lice per tank containing 35 fish, where point is mean infestation and lines indicate credible intervals (95%) in each dosage (low and high) and control. Figure 8 shows expected mean Caligus counts over the entire monitoring period (posterior median) for each life stage and treatment with 95% confidence intervals (margin of error) (Example 5, Experiment 1). Figure 9 shows expected Caligus reinfestation counts (posterior median, solid lines) for juveniles, adults and total lice with each treatment with 95% confidence intervals (shaded 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 pregnant females, females and adults for each treatment with 95% confidence intervals (shaded area). Black line means control (Example 5, Experiment 2). Figure 11 shows linear and quadratic coefficients for the dose-response model with 95% confidence intervals for each sample day (Example 5, Experiment 2). The quadratic effects are magnified (x 100) to make them visible in the same plot as the linear terms. See text for explanation.

Experimental part

Example 1 - Effect of carvacrol on copepodites of Caligus rogercresseyi

We have tested the compound carvacrol against copepodites of Caligus rogercresseyi by an in vitro method.

The objectives of this study are to; i) to determine whether carvacrol causes mortality of copepodites of Caligus rogercresseyi, and ii) to determine the LD50 (lethal dose causing 50% mortality) of carvacrol against copepodites of Caligus rogercresseyi in vitro.

Materials and methods

A study was conducted during January 2009 to determine the LD50 of carvacrol against copepodite stages of Caligus rogercresseyi in the experimental facilities of EWOS Innovation, Puerto Montt, Chile.

Copepodites for the study came from the Caligus rogercresseyi cultivation unit of the Fundacion Chile experimental facilities, located in Puerto Montt, Chile. The lice used in this study were taken after 9 generations of Caligus, which were grown under controlled conditions without exposure to anti-lice compounds.

Twenty-eight (28) petri dishes were filled with 40 ml of microfiltered (5 µm), aerated and sterilized seawater (with UV light at 70 uW/cm<3>). At the beginning of the study, 10 copepodites 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 ul/l. The control group comprised copepodites that were only exposed to seawater.

All petri dishes were placed in a constant temperature incubation chamber set at 14°C. The mortality of the copepodites was evaluated at 0, 1, 6, 16, 24 and 48 hours of incubation. The description of the different treatments is shown in table 1. The obtained data were analyzed using "R language" and its corresponding packages (http://www.r-project.org/). LD50 was modeled using a generalized linear model using a binomial model with logit link (http://cran.r-project.org/doc/manuals/fullrefman.pdf).

Results and discussion

The data show that the highest dose of carvacrol (20 pl/1) induces 100% mortality after 3 hours of exposure (Figure 1). Furthermore, it was observed that the copepodites became lethargic at doses above 7.5 ul/l after a few minutes of exposure to carvacrol (>2 min).

As a monoterpenoid, carvacrol is lipophilic and therefore we believe that a possible mechanism of action is passage of the cell wall and cytoplasmic membrane and the breaking up of structural layers of polysaccharides, fatty acids and phospholipids from the cells. No adverse effects of carvacrol have been described or reported for salmonids.

A time-dose response curve can be observed indicating that as the concentration of carvacrol increases, the mortality of the lice occurs earlier (Figure 2). All doses above 5 ul/l induce 100% mortality of the copepodites (Figure 2).

Results of the LD50 analyzes are shown in table 2. It can be observed that the LD50 for copepodites has a lower and upper confidence limit of 1.87 ul/l and 3.33 ul/l respectively after 48 hours of exposure to carvacrol with a calculated LD50 of 2.58 ul/l.

In the present LD50 investigation, it was possible to achieve narrow reliability limits, which were not far from the calculated LD50 (2.58 ul/l) that was achieved. This result increases the accuracy of the calculation.

The results showed that carvacrol induced a high mortality of copepodites of Caligus rogercresseyi at all exposure times tested in the present experiment (Table 2).

The following conclusions could be drawn from this experiment:

1. Carvacrol induces high mortality in copepodites of Caligus rogercresseyi at the concentrations tested in this experiment. 2. The calculated LD50 for carvacrol was 2.58 ul/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 has anti-lice activity against copedite stages of Caligus rogercresseyi. Therefore, the next step was to determine whether carvacrol has a similar activity against adult stages of C. rogercresseyi.

The purpose of this experiment is to determine the LD50 of carvacrol against adult Caligus rogercresseyi in vitro.

Materials and methods

A study was conducted during January 2009 to determine the LD50 of carvacrol against adult stages of Caligus rogercresseyi at the experimental facilities of EWOS Innovation, Puerto Montt, Chile.

Adult Caligus for the study came from the Caligus rogercresseyi Cultivation Unit of the Fundacion Chile, Experimental Facilities, located in Puerte Montt, Chile. The adults used in this study were obtained after 9 generations of Caligus grown under controlled conditions without any exposure to anti-lice compounds.

Twenty-four (28) petri dishes were filled with 40 ml of microfiltered (5 µm), aerated and sterilized seawater (with UV light at 70 uW/cm<3>). At the beginning of the study, 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 ul/l. The control group included adult Caligus kept in petri dishes with seawater.

All Petri dishes were placed in a constant temperature incubation chamber set at 14°C. Lice mortality was examined at 0, 1, 2, 3, 6, 12, 24, 36 and 48 hours of exposure to the active compound to be tested. The different treatments are shown in table 3.

The obtained data were analyzed using the statistical program SPSS, version 13.0. PROBIT was the statistical test used to determine the LD50.

Results and discussion

Similar to the results obtained in the previous study with copepodites, in the present experiment carvacrol induced a high mortality of the adult stages of Caligus rogercresseyi at all concentrations tested (Figure 3).

All doses of carvacrol tested in this bioassay killed 100% of the lice at 48 hours of exposure. The highest dose (20 µl/l) induced 100% mortality in 2 hours (Figure 3). Furthermore, a dose of 10 µl/l induced 100% mortality after 12 hours of exposure (Figure 3). Comparison of the results from this biological test with the previous study with copepodites shows that the adult stages of C. rogercresseyi may be more resistant to carvacrol than the juveniles.

A time-dose response can be observed indicating that as the concentration of carvacrol increases, mortality of adult lice occurs earlier (Figure 4).

The results of the LD50 analysis for each exposure time are shown in table 4. The calculations of LD50 doses vary widely and not systematically between the exposure times (table 4). After 12 hours of exposure with a smaller variability, it can be observed that the LD50 has a lower and upper confidence limit of 0.45Ml/l and 394.36 ul/l, respectively, with a calculated LD50 of 8.26 ul/l.

The following conclusions could be drawn from this experiment:

1. Carvacrol was shown to induce a high mortality rate in Caligus rogercresseyi adults at the concentrations tested in this experiment.

2. The calculated LD50 was 8.26 ul/l after 12 hours of exposure.

Example 3 - Effect of a fish feed containing carvacrol on lice

Consistent with the results obtained in Examples 1 and 2, carvacrol has a surprisingly high potential to reduce louse (C. rogercresseyi) infestation. No previous study has been carried out to test these phenolic compounds directly against lice, such as carvacrol and/or thymol. Furthermore, no previous study was carried out to investigate the effectiveness of carvacrol and thymol against lice when the fish is fed a food containing such compounds.

The purpose of this study is to investigate the effectiveness of carvacrol and thymol against lice {Caligus rogercresseyi) when feeding fish with a fish feed containing different doses of phenolic components, i.e. carvacrol and thymol.

Materials and methods

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

Four hundred and eighty (480) fish (-390 g/fish) were pit-tagged and distributed in twelve (12) containers of 0.35 m<3>, 40 fish/container, with filtered seawater (60 um). An acclimatization period of 7 days was carried out with a commercial lining. At day 8, the commercial feed was switched to the experimental feeds (see Table 5 below): a) control, b) 0.17% carvacrol+thymol in feed and c) 0.61% carvacrothymol in feed. Four replicates were used for each treatment. After 12 days of feeding the fish with the experimental feeds, the salmon were infested with copepodites of C. rogercresseyi (with an average infestation rate of 118 copepodites/fish) in 12 containers of 0.35 m3 with an open seawater flow system and an average temperature of about 13° C. Ten (10) days after the attack, 20 fish per container were sampled to count the number of attached lice on the fish, whereby the fish were previously anesthetized with AQUI-S (BAYER, Germany) in a bath for about 5 minutes. Lice were counted on live fish by 3 trained technicians without receiving the information about the distribution of the treatments in the various containers.

Results and discussion

After 10 days of infestation, a high reduction in total lice attached to the fish was achieved when the fish were fed a feed with carvacrol + thymol compared to the control feed (figure 6).

Table 6 gives calculations of the percentage reduction in total Caligustall for the carvacrol + thymol doses in comparison with the control.

For carvacrol+thymol, increasing the dose from 0.17 to 0.61 does not appear to have an additional effect on Caligus counts with reductions of 23.7% and 22.6%, respectively (Table 6). The tested product (both doses) had an average reduction of approximately 24% and a moderate probability (81%) supports a real effect (Table 7).

The following conclusions could be drawn from this experiment:

1. Carvacrol+thymol reduces the attack of Caligus rogercresseyi when the fish were treated with carvacrol and thymol added as a component of

the precomposition.

2. A dose of 0.17% of carvacrol+thymol (1:1) in the feed given for 22 days to Atlantic salmon (390 g/fish on average) reduced the infestation of lice (C. rogercresseyi) by about 24%.

From the preceding results, it can be concluded that carvacrol was effective in the control and reduction of parasites that cause disease in salmon.

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 preparation. The present invention thereby demonstrates the surprising potential of these components in the treatment and prevention of parasitic infections.

Example 4 - effect of carvacrol in the reduction of Caligus rogercresseyi infestation

The purpose of the study was to measure the efficacy of carvacrol in reducing Caligus rogercresseyi infestation when Atlantic salmon {Salmo salar) were fed a line with the compound in two different doses.

Initially, 528 Atlantic salmon were distributed in 4 tanks of 3 m3, with 132 fish per tank and a maximum density of 16 kg/m<3>, with a flow-through system and filtered seawater (60 µm). The fish were pit-tagged and weighed when distributed in each container. During 14 days, the fish recovered (7 days) and acclimatized (7 days) in the same containers fed with a commercial feed. Atlantic salmon were then challenged with copepodites of C. rogercresseyi (72 copepodites/fish) and 20 fish were sampled (basal sampling) to obtain liver, mucus (5ml), muscle (each fillet) and skin. The samples were stored and frozen at -80°C for further chemical analysis. After 10 days of infestation, all fish were sampled to tell the number of lice per fish (35 fish/container). The fish were then distributed into 15 containers, 0.35 m<3>/container, with 35 Atlantic salmon for each container. For 20 days, fish were fed three (3) diets: a) control diet, b) low dose of carvacrol in diet (30 mg carvacrol/kg diet) and c) high dose of carvacrol in diet (120 mg carvacrol/kg diet). The source of carvacrol used in the present experiment was a mixture of 50:50 wet weight/wet weight carvacrol+thymol. On the last day (day 20), the degree of infestation was determined by counting lice in all fish. The fish were individually weighed and samples of 5 fish per container were taken to obtain the liver, mucus (5 ml), muscle (each fillet) and skin. The samples were stored and frozen at - 80°C for further chemical analyzes of carvacrol concentration in each tissue.

Mortality was recorded daily. Environmental variables for each container were recorded four times per day, in particular dissolved oxygen in seawater (mg/l) and water temperature (°C).

Statistical analyses

Obtained data were analyzed using "R language" and its corresponding packages (http://www.r-project.org). To compare lice counts between different test feeds, the lice counts made in 35 individual sampled fish were converted to a total count per container by summing up the individual counts (given that the number of fish sampled is the same for all containers). This method is for unbound counts, such as random counts and they are assumed to follow the Poisson distribution. The lust counts can also be overdispersed, that is, the variance does not follow the standard Poisson distribution. This does not affect the coefficient calculations as such, but only their standard errors. Therefore, the model was a generalized linear model calculated with the glm function in "R language". The family is Poisson as overdispersion is modelled. The treatment calculations were based on posterior simulation (n=2500) with 95% confidence intervals as absolute and proportional to the reference level. Posterior estimates were averaged over the possible covariates and blocks (http://cran.r-project.org/doc/manuals/fullrefman.pdf).

Results

During the experiment, the water temperature in the containers varied between 17.8°C and 12.7°C and dissolved oxygen varied from 100% to 80% saturation. Pre-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 an average of 0.7%, with the lowest consumption observed in the first days of the experiment and the highest consumption towards the end of the study.

At the end of the experiment, the lice infestation in Atlantic salmon (S. salar) showed a high variability between the replicate containers within each treatment, which is not unusual for this type of study. Counts varied by approximately 2-fold within replicates (Table 9). Because of this wide variability, the calculations of dose effects showed wide confidence intervals (Table 9) (Figure 7). The results indicate that the probability of achieving a better response (reduction of louse infestation) with dosages of 120 (mg/kg) and 30 (mg/kg) of carvacrol is approximately 70% and 80%, respectively, compared to the control group. No significant statistical differences were observed between the treatments (p> 0.05).

Example 5 - effectiveness of carvacrol and thymol against Caligus rogercresseyi in rainbow trout (Onchorhynchus mykiss).

The effect of carvacrol and thymol against the different exchange steps in Caligus rogercresseyi has been investigated and the dosage of carvacrol and thymol in the feed was investigated to significantly reduce infestations of lice (Caligus rogercresseyi) in rainbow trout (O. mykiss).

Materials and methods

The effect of synthetic forms of carvacrol and thymol on the attack rate of Caligus rogercresseyi in rainbow trout (O. mykiss) was investigated in two experiments carried out in sea cages. The first experiment (Experiment 1) was carried out in a commercial marine facility located in the Xl region of Chile. In this experiment, three (3) dietary treatments were compared: 1) a commercial control feed (EWOS Silva 1500 and 2500) without any additive of any kind; 2) the same formulation as in 1, but with the addition of two functional components, nucleotides and a prebiotic (EWOS pre-harvest); and 3) the same treatment as in 2, but added 30 g/ton of feed (0.003%) of pure carvacrol and thymol (50:50 weight) (pre-harvest + new). The basic formulation of the compounds contains as main ingredients fish meal, poultry by-products, soybean products, sunflower meal, corn gluten, wheat, wheat gluten and a mixture of fish and vegetable oils (94.4% of the formulation).

Control, pre-harvest (PH) and pre-harvest + carvacrol and thymol (PH + new) compounds, were administered to 7, 3 and 4 cages, respectively. All cages belonged to the same location. Each cage contained approx. 50,000 fish. The experiment was carried out from 1 November 2009 to 28 February 2010. Fish weight increased from 1.7 kg to 2.5 kg during the experimental period. During this period, two pulses of dietary treatments were given to the trout. The first pulse was from 16 November to 7 December 2009, and the second pulse was from 20 January to 9 February 2010. Caligust counts were recorded on 10 fish per cage once a week during the two pulses. The measurements for salmon attack were carried out in two cages per treatment. The life forms of the lice recorded in each fish were total juveniles, total adults, gravid females and total number of lice.

The second experiment (Experiment 2) was carried out in January 2011 in the X region of Chile, at the test sea location of EWOS Innovation. This location has 16 cages of 15 x 15 m with an automatic feeding system. In this experiment, rainbow trout (O. mykiss) were used in all the cages. The average fish weight at the start of the experiment was 2.0 kg. Each cage contained approx. 10,000 fish. During this trial, four (4) dietary treatments were investigated. One of the feeds was a commercial control feed (EWOS Silva 2000) without any additives of any kind. Diets 2, 3 and 4 had the same basic formulation as the control feed, but added with a mixture of nucleotides, prebiotics, 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 basic formulation of these contained as main ingredients fish meal, poultry by-products, soya bean products, sunflower meal, maize gluten, wheat, wheat gluten and a mixture of fish and vegetable oil (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 experiments that were fed were 60, 90 and 240 g/ton of feed, respectively. Thus, as a percentage of the feed, pure carvacrol and thymol were added at 0.003, 0.006 and 0.012%, respectively.

EWOS Innovation's offshore location is located in a production area with natural Caligus infestation. To reduce and level the level of infestation in all fish before the start of the experimental period, a bath treatment with deltamethrin (Alphamax®, 0.2 ml/m<3>for 30 min) was carried out in all fish in a close tarpaulin system. After the bath treatment, an initial sampling of 30 fish per cage was carried out to count the lice infestation rate. The initial sample represents the basal attachment rate shortly after anti-parasite treatment and before the initial feeding of the experimental forages. All fish were anesthetized with Aqui-S and were weighed, marked and counted for lice, as standard procedure applied by EWOS Innovation. During each experimental period, 30 fish per cage were sampled on a weekly basis for lice counts. Louse life stages were recorded, including juvenile stages of copepodids, Chalimus I, II, II and IV and adult female stages, gravid females and males.

Statistical analyses

Caligust counts are expected to follow the Poisson distribution but that it is overdispersed. Fitting such multilevel overdispersed Poisson models is best performed with Markov chain Monte Carlo (MCMC) methods.

In Experiment 1, counts of different Caligus life stages were modeled as overdispersed Poisson and estimated with an MCMCglmm package of 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 (arbitrary effects). Similarly, a multi-level model for mean counts over the entire monitoring period was estimated using both cage and sampling data as arbitrary covariates. The model was estimated with MCMCglmm as described above.

Experiment 2 is a dose-response experiment and the potentially non-linear effect of dose was modeled with a second degree polynomial. Note: day of sampling was considered as a factor, which in practice means that the polynomial dose-response models between different trials are independent. The adapted model was used to calculate the development of expected lice numbers and the expected re-infestation counts (by subtracting the posterior distribution at day = 0). The latter is important since the initial lice numbers can vary between treatments due to the effectiveness of the bath treatment. The figures for different life stages typically show higher variation between the treatments than the total count. All statistical models were performed 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 entire monitoring period for each treatment with lice life stage are shown in Figure 8. The differences in the expected average counts exist between the treatments. There is a general trend for all life stages that PH treatment has a somewhat lower expected count than the control, and PH+ny treatment (with carvacrol and thymol) has an even lower expected count. For pregnant females and total lice, a strong evidence of lower counts can be claimed for PH+ny treatment (with carvacrol and thymol) compared to control. This is particularly relevant and interesting because it is obvious that pregnant females are an important triggering factor in the development of a louse population. A comparable effect of lower counts has also been observed for PH treatment when considering only pregnant females. There is a difference between PH and PH+ny treatment as the PH+ny diet

(with carvacrol and thymol) seems to also affect young animals and adults, while the PH diet only affects pregnant females.

Experiment 2

At the start of the experiment, the attack by lice { Caligus rogercresseyi) differed between the different experimental groups (Table 10)

Reinfection was calculated from posterior distributions of the model by subtracting the posterior distributions of the control and experimental treatments at day=0 from the distributions on the other days. Re-attachment counts can be negative if the lice count for a specific life stage is reduced from the initial count. The results are shown in Figures 9 and 10. These graphs show that the fish fed with carvacrol and thymol have a lower reattachment rate for juveniles, adults and total lice in a dose dependent trend (Figure 9). An inclusion of as low as 0.003% carvacrol and thymol in the feed was surprisingly effective for the reattachment of lice despite the comparably low dosage.

Although higher doses of carvacrol and thymol had a stronger effect than the lower doses tested. A similar effect is observed for pregnant females, females and males (Figure 10).

To better understand and see the effects of the dosage of carvacrol and thymol on the different life stages of the louse, the linear and quadratic coefficients of the dose response model for each sampling day are plotted against their 95% confidence intervals (Figure 11). This figure requires some explanation. The linear and quadratic coefficients describe the shape of the dose effect and the confidence intervals show what certain such effects are like. Therefore, we look for statistically significant coefficients that have 95% confidence intervals that do not cross the zero line marked on the graph.

On day 7, some linear coefficients can be seen for Ch l&ll, pregnant females, females, males and total number of lice, and all are negative, meaning that carvacrol and thymol linearly and significantly reduce the re-attachment of these life stages. The linear coefficients imply that the dosage has a linear effect on the lice numbers. The only quadratic coefficient that is significant is the positive coefficient for pregnant females, but many other life stages that have significant linear coefficient trends also appear to have a positive quadratic coefficient. By placing a negative linear and positive quadratic coefficient together, this means that the dosage initially has a reducing effect on the number of lice, but that the effect decreases with higher dosages. At day 14, the picture is very similar to day 7. Several quadratic coefficients can now be considered significant, i.e. they confirm the type of dosage effect explained above for day 7. A similar effect on copepodids is also explained. There now appears to be a positive linear coefficient with a negative quadratic coefficient for Ch lll&IV, which indicates that the initial increase in dosage has little or no effect and only the highest dosages can be effective for this life phase. At day 21, the only significant effects are in negative linear and positive quadratic coefficients for pregnant females and the opposite for Ch lll&IV.

Conclusions

The following conclusions are reached from this experiment:

- Dietary addition of carvacrol and thymol significantly reduces the reattachment of different life stages of Caligus rogercresseyi during a period of 3-4 weeks in rainbow trout. - At very low and higher dosages of carvacrol and thymol (0.003 to 0.012%), the effect was more obvious on pregnant females, while a dosage of 0.012% was necessary to also reduce attachment to young animals.

Thus, with the present invention it has also been shown for the first time that there are clear differences in the effectiveness of thymol and carvacrol on different life stages of ectoparasitic copepod louse, depending both on the period of application and concentration of the active compounds. Already comparable low dosages are effective for pregnant females while higher dosages seem to be necessary to control juveniles. There is also a practical relevance for the use of the active compounds in the treatment of lice infestations. It suggests that carvacrol and thymol can be used in the regulation of lice where one specifically directs the attack and the fight against pregnant females in specific periods by using low concentrations of the active compounds.

The effectiveness of carvacrol and thymol in the treatment of lice is increased by the presence of immunostimulants in the feed as shown in Example 5. Addition of immunostimulants is not limited to non-specific immunostimulants, but can also be chosen from among specific immunostimulants.

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, where the active compounds are preferably added to the feed and consumed by the fish on a daily basis. It is preferred that the fish are fed the diet for at least 7 days, more preferably for 3-4 weeks. However, longer periods exceeding 28 days may also be applied.

It is assumed that the same effects as shown for 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 must be recognized that embodiments of the invention described in the foregoing can be modified without deviating from the scope of the invention.

Definitions used in the application

Carvacrol is the compound 5-isopropyl-2-methylphenol (C6H3CH3(OH)(C3H7)), and is a monoterpenoid phenol. It has a characteristically strong, warm smell of oregano and a pizza-like taste. It is present in the essential oil of Origanum vulgare, oil of thyme, oil obtained from cress, and wild bergamot.

Thymol is the compound 5-isopropyl-2-methylphenol, and is a monoterpenoid phenol derivative of cymene, isomeric with carvacrol, found in thyme, and is extracted as a white crystalline substance with a good aromatic odor and strong antiseptic properties.

Prebiotics are pre-ingredients that stimulate the weight and/or activity of bacteria in the digestive system that are useful for health.

Immunostimulants are substances (drugs and nutritional products) that stimulate the immune system by inducing activation or increasing activity of some of its components. They can be non-specific or specific immunostimulants.

Conventional pre-ingredients are pre-ingredients that are usually used in pre-compositions for a specific animal species such as lipids, proteins, vitamins, carbohydrates, minerals etc.

Nucleotides include any known phosphoric ester of a nucleoside such as AMP, GMP,

UMP, CMP, UMP.

Claims (48)

1. 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 attachment in a fish. 2. The compound or composition according to claim 1, wherein the ectoparasitic copepod is a louse. 3. Compound or composition in accordance with claim 2, wherein the louse is selected from Lepeophtheirus salmonis, Caligus elongatus and Caligus rogercresseyi. 4. Compound or composition in accordance with claim 1, where the ratio of carvacroctymol is in the range 1:3 to 3:1, more preferably 1:2 to 2:1, most preferably approx. 1:1, based on weight. 5. Compound or composition in accordance with one of claims 1-4, where said fish is a salmon fish. 6. Compound or composition in accordance with claim 5, wherein said fish is selected from the group consisting of Atlantic salmon (Salmo salar), rainbow trout (Onchorhynkuss mykiss), Coho salmon (Oncorhynchus kisutch) and arctic char (Salvelinus alpinus). 7. Compound or composition in accordance with one of the preceding requirements for reducing the total number of adult lice stages attacked or re-infested the fish. 8. Compound or composition in accordance with one of the preceding claims, where carvacrol and/or thymol is added to a pre-composition in a total concentration of between 0.001% to 0.8% of the feed, preferably 0.003% to 0.6% and more preferably between 0.003 and 0.012% by weight of the feed. 9. Compound or composition according to claim 1, wherein the total concentration of carvacrol and/or thymol is 30 mg/kg or more, preferably 45 mg/kg for or more, more preferably at least 100 mg/kg and most preferably at least 120 mg/kg for. 10. A compound or composition according to claim 8 or 9, wherein carvacrol and thymol are in a ratio of 1:1. 11. Compound or composition in accordance with one of the preceding requirements where the feed includes other conventional ingredients in the fish feed. 12. Compound or composition in accordance with one of the preceding claims, wherein the feed further comprises one or more of the following ingredients selected from the group of nucleotides, prebiotics, vitamin C and immunostimulants, which may be specific or non-specific. 13. Compound or composition in accordance with one of the preceding claims, where the compound or composition is supplied to fish for at least 7 days, more preferably at least 21 days and most preferably at least 28 days. 14. Compound or composition in accordance with one of the preceding claims, where the treatment with the active compounds is combined with other chemical and medicinal agents against lice. 15. Compound or composition in accordance with one of the preceding claims, where the active compounds are specifically used to combat certain life forms of lice. 16. Compound or composition in accordance with claim 15, where the active compounds are used to reduce the attachment of pregnant female lice to the fish. 17. Compound or composition in accordance with claim 13, wherein the fish are continuously fed feed comprising the active compounds as part of their daily diet. 18. Compound or composition in accordance with claim 1, where the active compound or compounds are applied in the form of a water bath treatment. 19. Method for killing, controlling or controlling ectoparasitic copepods, where the method comprises applying carvacrol and/or thymol or a composition comprising one or both of the active compounds to kill the lice, to make them drowsy or to prevent attachment to fish . 20. Method according to claim 19, wherein said ectoparasitic copepod is a louse. 21. Method in accordance with claim 20, where said louse is Lepeophtheirus salmonis, Caligus elongatus or Caligus rogercresseyi. 22. Method according to one of the preceding claims, wherein said fish is a salmon fish, preferably selected from the group consisting of Atlantic salmon (Salmo salar), rainbow trout (Onchorhynkuss mykiss), Coho salmon (Oncorhynchus kisutch) and arctic char ( Salvelinus alpinus). 23. Method in accordance with claim 19, where said active compound, mixture of compounds or composition is applied to a locus to be protected from the parasites. 24. Method in accordance with claim 23, where said locus is an aqueous environment and where the active compound or composition comprising the active compound is applied directly to the aqueous locus. 25. Method in accordance with claim 24, where said locus contains fish and where the louse is located on said fish or said fish are susceptible to attack or infection by said louse, where the active compound or mixture of compounds is applied directly to the fish, preferably with topical or oral application. 26. Method in accordance with claim 19, where the active compound or mixture of compounds is applied to the fish as a component of the feed that is fed to the fish. 27. Method according to one of claims 21-26, where the carvacrothymol ratio is in the range 1:3 to 3:1, more preferably 1:2 to 2:1, and most preferably approx. 1:1, based on weight. 28. Method according to one of the preceding claims, where one or more of the active compounds or a composition comprising the active compounds is supplied to the fish as part of the feed for at least 7 days, more preferably for at least 21 days and most preferably at least 28 days. 29. Use of carvacrol and/or thymol for the preparation of a pharmaceutical composition for the treatment and/or prophylaxis of ectoparasitic copepod infection and/or attachment in a fish. 30. Use in accordance with claim 29, wherein said ectoparasitic copepod is a louse. 31. Use in accordance with claim 30, where said louse is selected from among Lepeophtheirus salmonis, Caligus elongatus and Caligus rogercresseyi. 32. Use in accordance with claim 29, where the carvacrol/thymol ratio is in the range 1:3 to 3:1, more preferably 1:2 to 2:1, and most preferably approx. 1:1, based on weight. 33. Application in accordance with one of claims 29-32, where said fish is a salmon fish. 34. Use in accordance with claim 33, where said fish is selected from the group consisting of Atlantic salmon (Salmo salar), rainbow trout (Onchorhynkuss mykiss), Coho salmon (Oncorhynchus kisutch) and arctic char (Salvelinus alpinus). 35. Application in accordance with one of the preceding requirements for reducing the total number of lice forms that attach or reattach to the fish. 36. Use in accordance with one of the preceding claims, where the pharmaceutical composition comprising carvacrol and/or thymol is added to a pre-composition in a total concentration of carvacrol and/or thymol from 0.001% to 0.8% of the feed, preferably 0.003% to 0.6% and more preferably from 0.003 and 0.012% by weight of the feed. 37. Use in accordance with claim 29, where said pharmaceutical composition comprising carvacrol and/or thymol is added to a pre-composition, which has a total concentration of carvacrol and/or thymol of at least 30 mg/kg for, preferably at least 45 mg/kg for, more preferably at least 100 mg/kg and most preferably at least 120 mg/kg for. 38. Use in accordance with claim 36 or 37, where said carvacrol and thymol are in a ratio of 1:1. 39. Application in accordance with one of the preceding claims, where the feed includes other conventional ingredients in the fish feed. 40. Use in accordance with one of the preceding claims, where the feed further comprises one or more of the following ingredients selected from the group consisting of nucleotides, prebiotics, vitamin C and immunostimulants, which may be specific or non-specific. 41. Use in accordance with one of the preceding claims, where the compound or composition is supplied to fish for at least 7 days, more preferably at least 21 days and most preferably at least 28 days. 42. Application in accordance with one of the preceding claims, where the treatment with the active compounds is combined with other chemical and medicinal agents against lice. 43. Use in accordance with one of the preceding claims, where the active compounds are specifically used to combat certain life forms of lice. 44. Use in accordance with claim 43, where the active compounds are used to reduce the attachment of pregnant female lice to the fish. 45. Use in accordance with claim 41, where the fish are continuously fed a feed comprising the active compounds as part of their daily feed. 46. Application in accordance with claim 29, where the pharmaceutical composition is applied in the form of a water bath treatment. 47. Feed for fish, comprising carvacrol and/or thymol where the total concentration of carvacrol and/or thymol is at least 45 mg/kg feed, more preferably at least 100 mg/kg and most preferably at least 120 mg/kg feed. 48. For in accordance with claim 47, where the ratio carvacrol:thymol is in the range 1:3 to 3:1, more preferably 1:2 to 2:1, most preferably approx. 1:1 based on weight.