Field of the invention

The present invention relates to Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins for use in the prevention and/or treatment of ectoparasitic infection or infestation in fish. The present invention also relates to a method for the manufacture of a fish feed comprising Quillaja Saponaria saponins.

Background of the invention

Ectoparasitic copepods are copepods that feed on the mucus, epidermal tissue and/or blood of fish. Ectoparasitic copepods include sea lice, which includes copepods within the family Caligidae. This family includes around 162

Lepeophtheirus and 268 Caligus species.

Ectoparasitic copepods such as sea lice may cause physical and enzymatic damage at the site of attachment on the fish which results in abrasion-like lesions that may vary in their nature and severity. A sea lice infection itself may cause a generalized chronic stress response in fish since feeding and attachment cause changes in the mucus consistency and damage the epithelium resulting in the loss of blood and fluids, electrolyte changes and Cortisol release. These changes can decrease the immune response and affect negatively the feed intake leading to a significant reduction of growth and performance. Sea lice infections also make the fish susceptible to other diseases and may as a secondary infection be a possible vector for other pathogens like salmon alphe virus (Pancreatic disease), Infectious Salmon Anamis virus (ISAv) and Piscirickettsia salmonis. The degree of damage is also dependent on the species of sea lice, the developmental stages of the sea lice present, and the number of sea lice on the fish. Economic losses caused by ectoparasitic copepods such as sea lice can be tremendous and significant research effort has been dedicated to treat, control and prevent such diseases in aquaculture production. Moreover, ectoparasitic infestations/infections in farmed fish are not only harmful for the cultivated fish, they also represent a constant and severe thread for wild populations, which can be infected by the farmed fish. This is another reason why an effective control and treatment of ectoparasitic copepods and other infectious diseases is of great importance. Ectoparasitic copepods of the family Caligidae was first identified in Chile during 1997 in Atlantic salmon. From 1997 and until a few years ago sea lice of the family Lepeophtheirus and Caligus was treated with emamectin benzoate.

Unfortunately, the efficiency of emamectin benzoate has diminished significantly due to the excessive use and lack of rotation with other fish-specific antiparasitic compounds. Such compounds include organophosphates, pyrethroids, hydrogen peroxides and chitin synthesis inhibitors. At present, the strategy applied to decrease infestations/infections with sea lice is a combination of emamectin benzoate and newer pesticides such as Deltamethrin, Cypermethrin, Azametofos and Hydrogen peroxide administrated by immersion. The effectiveness of this strategy however, has not reached the level expected and infestations/infections caused by sea lice still poses a huge problem to the farming industry.

Newer strategies to decrease infestations with ectoparasitic copepods includes immunestimulants such as beta-glucans, essential oils (carvacrol, thymol and their combinations), and other nutraceuticals such as nucleotides and mannan- oligosaccharides, organic minerals like zinc and therapeutic levels of vitamin C. Such strategies may be applied alone or in combination with the chemical strategies mentioned above. Fish-specific antiparasitic compounds, such as compounds capable of preventing and/or treating ectoparasitic copepods like sea lice, should preferably be non-toxic for the fish in the applied concentrations and from an environmental point of view, the compounds should be fast and easily degradable in the natural environment without any unfavourable and harmful accumulation in the nutritional chain. When supplied with the diet, such compounds should either be degraded or excreted by the organism after a reasonable time period . This is especially important since farmed fish will eventually be consumed by humans.

In Chile over the last couple of years, the economic losses recorded due to the management of sea lice outbreaks has become one of the most important items in the Salmon and Trout production costs. In this scenario the development of alternative treatments for the control of ectoparasitic copepod

infestations/infections such as infestations/infections caused by sea lice, is urgent. In this respect, it would be especially advantageous if such treatment could be included in the fish feed.

Summary of the invention

Thus, an object of the present invention relates to a compound or composition for use in the prevention and/or treatment of ectoparasitic infestations or infections in fish.

In particular, it may be seen as an object of the present invention to provide a compound or composition that solves the above mentioned problems of the prior art - e.g. by being essentially non-toxic for the fish in the applied concentrations when incorporated into a fish feed.

Thus, one aspect of the invention relates to Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins for use in the prevention and/or treatment of ectoparasitic infection or infestation in fish.

Another aspect of the present invention relates to the use of Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins for the manufacture of a medicament for the treatment and/or prevention of ectoparasitic copepod infection or infestation in fish.

Yet another aspect of the present invention is to provide a method of treating and/or preventing ectoparasitic copepod infection or infestation in fish, said method comprising oral administering to said fish Quillaja Saponaria or a composition comprising Quillaja Saponaria saponins.

Still another aspect of the present invention is to provide a method for the manufacture of a fish feed, comprising the steps of combining Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins with approved and/or authorised fish feed ingredients and obtaining a fish feed comprising Quillaja Saponaria saponins.

Description of the figures

Figure 1 shows the toxicity of Quillaja saponaria saponins on Caligus. The dose "ppm" in figure 1 refers to experiments conducted with Quillaja high extract which comprises 20% Quillaja saponaria saponins - the dose thus corresponds to 0, 0.1, 10 and 100 ppm Quillaja saponaria saponins.

Figure 2 shows the effect of Quillaja saponaria saponins on the infection rate with Caligus in Atlantic salmon (Salmo salar).

Figure 3A-3C shows the SGR (specific growth rate); SFR (specific feed rate) and FCR (feed conversion rate) between two groups.

Figure 4 shows the chemical formula of s triterpenoid saponin.

Figure 5 shows that Quillaja saponaria saponins reduce the infestation rate with Caligus under commercial conditions. Fish from a seawater site were divided in two groups; unit 1 that was fed with a Control Diet and unit 2, fed with a Test Diet added, which corresponds to the Control Diet with the addition of Quillaja saponaria saponins. At the start of the feeding strategy and 33 days after the starting of the feeding regimen, the number of Caligus attached to the skin of fish and those parasites present in the bins of counting were recorded. The counting considered the following life cycle stages: Chalimus, adult females and males and gravid females. The infestation rate calculated is expressed as a percentage. The values represent the mean of 5 cages per condition ± SE. (*) statistically significant difference. One Way ANOVA analysis at P<0.050. The present invention will now be described in more detail in the following.

Detailed description of the invention

The present inventors have surprisingly discovered that saponins from Quillaja saponaria in vitro presents a high toxicity to Caligus. Moreover in vivo studies performed by the present inventors show a significant reduction of Caligus in Atlantic salmon fed with a feed comprising Quillaja saponaria saponins compared to a control group fed with a traditional feed . Thus, one embodiment the present invention pertains to Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins for use in the prevention and/or treatment of ectoparasitic infection or infestation in fish.

In another embodiment, the present invention pertains to the use of Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins for the manufacture of a medicament for the treatment and/or prevention of ectoparasitic copepod infection or infestation in fish.

A further embodiment of the present invention pertains to a method of treating and/or preventing ectoparasitic copepod infection or infestation in fish, said method comprising administering to said fish Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins.

It is to be understood that the term "prevention" also covers prophylactic use of the compound, the medicament or method disclosed above.

Quillaja Saponaria saponins

Quillaja (Quillaja saponaria) is a tree native to Chile. The inner bark of Quillaja saponaria can be reduced to powder and employed as a substitute for soap due to the presence of saponins. Thus, Quillaja Saponaria saponins may also be referred to as inner bark of Quillaja saponaria. They may be provided as a powder or liquid, and may be in a purified, partly purified or native state. Saponins are mainly produced by plants, but also by lower marine animals and some bacteria (Yoshiki et al., 1998; Riguera, 1997). Saponins derive their name from their ability to dissolve in water to form colloidal solutions that produce foam upon shaking. These molecules consists of a group of glycosides with surface active properties that distinguish these compounds from other glycosides. Quillaja Saponaria saponins consist of a sugar moiety, usually comprising glucose, galactose, glucuronic acid, xylose, rhamnose or methyl pentose, glycosidically linked to a hydrophobic aglycone also refeered to as a sapogenin. The sapogenin can be a triterpenoid or a steroid or derivative thereof (Francis et al., 2005).

Saponins from Quillaja saponaria belongs to the group of triterpenoid saponins and will in the following be termed Quillaia saponins or Quillaja Saponaria saponins.

Although saponins play a key role in the defence response of plants against the insects attack, it is believed that saponins exert a negative effect on animals if present in an animal diet in moderate to high concentrations (Cheeke, 1996). In fact dietary saponins derived from different plants have been considered as the etiological factor of a significant reduction of feed intake and impairment of productive performance of salmonid fish (Newinger, 1994;Bureau et al., 1998). Thus, in one embodiment of the present invention the Quillaja Saponaria saponins are terpenoid saponins. Terpenoid saponins may be selected from the group consisting of monoterpenoid saponins, diterpenoid saponins and triterpenoid saponins. In a preferred embodiment the terpenoid saponins are triterpenoid saponins.

The chemical formula of a triterpenoid saponin is shown in figure 4. Ectoparasitic copepod

As mentioned above ectoparasitic copepods are copepods that feed on the mucus, epidermal tissue and/or blood of fish.

Sea lice are ectoparasitic copepods and belong to the family Caligidae. This family includes around 162 Lepeophtheirus and 268 Caligus species. Thus, in an embodiment of the present invention the ectoparasitic copepod may be sea lice. In a further embodiment the sea lice may be of the family Caligidae. In a further embodiment the sea lice may be of the species Lepeophtheirus or Caligus and more specifically they may be selected from the group consisting of Lepeophtheirus salmonis, Caligus elongatus and Caligus rogercresseyi.

Sea lice of different life stages has been found on fish - such life stages include but is not limited to Chalimus I, Chalimus II, Chalimus III, Chalimus IV, pre- adults, adult sea lice and gravid females.

Many ectoparasites such as sea lice are specific with regards to host genera. For example L. salmonis has a high specificity for salmonids, including the widely farmed Atlantic salmon (Salmo salar). L. salmonis may also parasitize other salmonids to varying degrees, including brown trout (sea trout: Salmo trutta), arctic char (Salvelinus alpinus) and all species of Pacific salmon.

Caligus rogercresseyi has been recognized as the major parasite of farmed salmon in Chile. It was first reported in 1997 in Atlantic salmon. Currently the most susceptible reported hosts are Atlantic salmon and rainbow trout. Caligus rogercresseyi also parasitizes wild species, being the Patagonian blenny

(Eleginops maclovinus) one of the most important due to its role as reservoir and spreader of Caligus to farmed salmonid fish.

Fish

Infestations or infections with sea lice occur in fish.

In the present context the terms "infestation" and "infection" are used herein interchangeably since some scientific articles written in English use the terms "infection" or "infestation" to develop a matter that explains diseases caused by parasites. Thus, in an embodiment of the present invention the fish is of the family Salmonidae or may be of the subfamily Salmoninae. In a preferred embodiment the fish may be of a genus selected from the group consisting of Salmo, Oncorhynchus and Salvelinus.

If the fish is of the genus Salmo the fish may be selected from the group consisting of Atlantic salmon {Salma salar L), Adriatic trout {Salmo obtusirostris), Flathead trout (Salmo platycephalus), Marble trout (Salmo marmoratus), Ohrid trout (Salmo letnica) and Sevan trout (Salmo ischchan). Salmon is produced by mixing the eggs from one female salmon with the milt of one male salmon for fertilization. Once the eggs have hardened, they are transported to a hatchery for incubation . Each egg batch is incubated separately in Heath trays for approximately 5-6 weeks until they reach the eyed stage and will hatch after a total of approximately 9 weeks. Upon hatch, the alevins or sac fry will continue to develop using the nutrients in their yolk sacs. When their yolk sacs are almost consumed, the young fry are ready to feed . Once the young fry are onto feed they grow quickly over the next 6 to 12 months in freshwater. The fry grow to become parr/fingerlings and finally undergo developmental changes that prepare them for life in saltwater as smolts. Smolts ready for saltwater are transported from the hatchery to the marine farms, where they are transferred to the net pens to begin the seawater/saltwater phase of their life cycle. Smolts entered at the sea sites adjust to the saltwater environment and begin feeding. They are fed a high quality diet specially formulated to match the optimal seasonal and life stage requirements of salmon in the wild . Once onto feed, smolts grow quickly in seawater/saltwater, reaching a kilogram in size in 6 to 8 months.

Thus, in an embodiment of the present invention the fish may be selected from the group consisting of smolts and adult fish. Therefore, the compound, composition and/or feed of the present invention may be administered to fish in the freshwater phase or the seawater/saltwater phase. The invention may be useful to control e.g. parasitic infection with sea lice during the

seawater/saltwater stage. However, it may be preferred that the compound, composition and/or feed of the present invention is administered during the last period of the freshwater phase with the objective to prepare fish before the transfer to seawater/saltwater i.e. as a prevention of a potential ectoparasitic infection or infestation.

If the fish is of the genus Oncorhynchus the fish may be selected from the group consisting of Chinook salmon (Oncorhynchus tshawytscha), Chum salmon

(Oncorhynchus keta), Coho salmon (Oncorhynchus kisutch), Pink salmon (Oncorhynchus gorbuscha), Sockeye salmon (Oncorhynchus nerka), Masu salmon (Oncorhynchus masou), Biwa trout (Oncorhynchus rhodurus), Cutthroat trout (Oncorhynchus clarkii), Rainbow trout (Oncorhynchus mykiss) and Mexican Golden Trout (Oncorhynchus chrysogaster).

If the fish is of the genus Salvelinus the fish may be Arctic char (Salvelinus alp in us).

In a particular preferred embodiment, the fish may be selected from the group consisting of Atlantic salmon (Salma salar L.) and Rainbow trout (Oncorhynchus mykiss) .

Administration and formulation

One preferred administration form of the Quillaja Saponaria saponins, the composition comprising Quillaja Saponaria saponins and/or the medicament comprising Quillaja Saponaria saponins is oral dosing. The dosing may preferably be in the form of a feed comprising Quillaja Saponaria saponins - i.e. a fish feed .

In one embodiment of the present invention the Quillaja Saponaria saponins, the composition comprising Quillaja Saponaria saponins and/or the medicament comprising Quillaja Saponaria saponins may be part of a fish feed or may be formulated as a fish feed . Such feed may be selected from the group consisting of pressed fish feed, pelleted fish feed, expanded fish feed, extruded fish feed and wet semimoist feed . Which of the feed types to choose depends on the fish species, the maturity of the fish and to which environment the feed is to be applied .

In a particular embodiment the compositon and/or fish feed of the present invention comprise Quillaja Saponaria saponins in combination with approved and/or authorised fish feed ingredients. The approved fish feed ingredients may selected from the group consisting of a carbohydrate source, a protein source, a lipid source, ash, water and any combinations thereof. In the context of the present application the terms "approved fish feed

ingredients" and "authorised fish feed ingredients" are used interchangeably. In the context of the present application the terms "approved fish feed ingredients" and "authorised fish feed ingredients" have the following meaning : fish feed ingredients covering feed materials such as products of vegetable or animal origin, whose principal purpose is to meet animals' nutritional needs, in their natural state, fresh or preserved, and products derived from the industrial processing thereof, and organic or inorganic substances, whether or not containing feed additives, which are intended for use in oral animal-feeding either directly as such, or after processing, or in the preparation of compound feed, or as carrier of premixtures. In this context, "carrier" can be a substance used to dissolve, dilute, disperse or otherwise physically modify a feed additive in order to facilitate its handling, application or use without altering its technological function and without exerting any technological effect itself.

Typically the protein source may constitute from 25-55% (w/w) of the

composition and/or fish feed, such as from 26-54% (w/w), e.g . from 27-53% (w/w), such as from 28-52% (w/w), e.g. from 27-51% (w/w), such as from 28- 50% (w/w), e.g. from 29-49% (w/w), such as from 30-48% (w/w), e.g. from 31- 47% (w/w), such as from 32-46% (w/w), e.g. from 33-45% (w/w), such as from 34-44% (w/w), e.g. from 35-43% (w/w), such as from 36-42% (w/w), such as from 37-41% (w/w), e.g . from 38-40% (w/w), such as from 39-40% (w/w), preferably in the range from 30-55% (w/w). The carbohydrate source may constitute from 5-25% (w/w) of the composition and/or fish feed, such as from 6-24% (w/w), e.g. from 7-23% (w/w), such as from 8-24% (w/w), e.g. from 9-23% (w/w), such as from 10-24% (w/w), e.g. from 11-23% (w/w), such as from 12- 22% (w/w), e.g. from 13-21% (w/w), such as from 14-20% (w/w), e.g. from 15-19% (w/w), such as from 16-18% (w/w), e.g. from 17-19% (w/w), preferably in the range from 10-15% (w/w).

The lipid source may constitute from 14-40% (w/w) of the composition and/or fish feed, such as from 15-39% (w/w), e.g. from 16-38% (w/w), e.g. 17-37% (w/w), such as from 18-36% (w/w), e.g. from 19-35% (w/w), such as from 20-36% (w/w), e.g. from 21-35% (w/w), such as from 22-36% (w/w), e.g. from 23-35% (w/w), such as from 24-34% (w/w), e.g. from 25-33%, such as from 26-32% (w/w), e.g. from 27-33%, such as from 28-32% (w/w), e.g. from 29-31%, such as from 30-31% (w/w), preferably in the range from 25-40% (w/w). Typical protein sources may be processed animal protein or plant protein. The processed animal protein may be selected from the group consisting of fish meal, blood meal, pork meal, feather meal, meat meal, bone meal and any

combinations thereof whereas the plant protein may be selected from the group consisting of soybean, peanut, corn, gluten, raps, lupine, sunflower, wheat, sorghum, pea, oat and any combination thereof.

The carbohydrate sources may be selected from the group consisting of corn, wheat, beans, peas, tapioca, potato starch, sorghum, oat and any combination thereof.

The lipid sources may selected from the group consisting of fish oils, plant oils, poultry oil and any combinations thereof and the plant oil may be selected from the group consisting of soybean, rapeseed, sunflower, linseed oil and any combinations thereof.

In a preferred embodiment the fish feed may comprises an ingredient selected from the group consisting of nucleotides, prebiotics, probiotics, vitamins, minerals, antioxidants, liposoluble and hydrosoluble molecules extracted from vegetables, liposoluble and hydrosoluble molecules extracted from marine sources, immune stimulants and any combinations thereof. Which ingredient(s) to choose depends on the fish species, the maturity of the fish and to which environment the feed is to be applied .

As can be seen in Example 1 and figure 1 the present inventors shows that the mortality of Caligus presented a dose response pattern achieving a 50, 85 and 100% mortality at 0.1, 10 and 100 ppm of Quillaja Saponaria saponins

respectively. In Example 2 and figure 2 the present inventors showed that feeding Atlantic salmon comprising 200 ppm of Quillaja Saponaria saponins lead to a 44.3% reduction of the number of Caligus per fish compared with the group of fish fed with a control diet. Moreover it can be seen from figures 3A-3C that there are no statistically significant differences on specific growth rate (SGR), specific feed rate (SFR) and feed conversion rate (FCR) between the groups in Example 2.

Thus, in a preferred embodiment of the present invention the composition and/or fish feed may comprise at least 0.1 ppm Quillaja Saponaria saponins, e.g. at least 0.2 ppm Quillaja Saponaria saponins, such as at least 0.3 ppm Quillaja Saponaria saponins, e.g . at least 0.4 ppm Quillaja Saponaria saponins, such as at least 0.5 ppm Quillaja Saponaria saponins, e.g. at least 0.6 ppm Quillaja Saponaria saponins, such as at least 0.7 ppm Quillaja Saponaria saponins, e.g. at least 0.8 ppm Quillaja Saponaria saponins, such as at least 0.9 ppm Quillaja Saponaria saponins, e.g . at least 1 ppm Quillaja Saponaria saponins, such as at least 5 ppm Quillaja Saponaria saponins, e.g. at least 10 ppm Quillaja Saponaria saponins, such as at least 50 ppm Quillaja Saponaria saponins, e.g. at least 100 ppm

Quillaja Saponaria saponins, such as at least 200 ppm Quillaja Saponaria saponins, e.g. in the range from 0.1 - 200 ppm Quillaja Saponaria saponins, such as in the range from 0.2 - 180 ppm Quillaja Saponaria saponins, e.g. in the range from 0.3 - 170 ppm Quillaja Saponaria saponins, such in the range from 0.4 - 160 ppm Quillaja Saponaria saponins, e.g. in the range from 0.5 - 150 ppm Quillaja Saponaria saponins, such in the range from 0.6 - 140 ppm Quillaja Saponaria saponins, e.g. in the range from 0.7 - 130 ppm Quillaja Saponaria saponins, such in the range from 0.8 - 120 ppm Quillaja Saponaria saponins, e.g. in the range from 0.9 - 110 ppm Quillaja Saponaria saponins, such in the range from 0.8 - 100 ppm Quillaja Saponaria saponins, e.g. in the range from 0.9 - 90 ppm Quillaja Saponaria saponins, such in the range from 1 - 80 ppm Quillaja Saponaria saponins, e.g. in the range from 5 - 70 ppm Quillaja Saponaria saponins, such in the range from 10 - 60 ppm Quillaja Saponaria saponins, e.g. in the range from 20 - 50 ppm Quillaja Saponaria saponins, such in the range from 30 - 40 ppm Quillaja Saponaria saponins. Saponins may be measured by means of Ultra Performance Liquid

Chromatography i.e. UPLC (a modified HPLC method).

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise at least 0.1 ppm Quillaja Saponaria saponins.

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 0.1 - 200 ppm Quillaja Saponaria saponins.

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 0.4 - 160 ppm Quillaja Saponaria saponins. In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 0.8 - 120 ppm Quillaja Saponaria saponins.

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 1 - 80 ppm Quillaja Saponaria saponins.

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 5 - 70 ppm Quillaja Saponaria saponins.

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 10 - 60 ppm Quillaja Saponaria saponins.

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 20 - 50 ppm Quillaja Saponaria saponins. In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 30 - 40 ppm Quillaja Saponaria saponins.

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 0,1 ppm Quillaja Saponaria saponins.

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 10 ppm Quillaja Saponaria saponins.

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 100 ppm Quillaja Saponaria saponins.

In a preferred embodiment the composition and/or fish feed comprise the following constituents in the following amounts: (i) protein source constitutes from 25-55% (w/w) of the composition and/or fish feed, (ii) carbohydrate source constitutes from 5-25% (w/w) of the composition and/or fish feed, (iii) lipid source constitutes from 14-40% (w/w) of the composition and/or fish feed (iv) the composition and/or fish feed comprise 200 ppm Quillaja Saponaria saponins. In order to obtain a satisfactory result, it may be preferred that the fish are continuously fed Quillaja Saponaria saponins, a composition comprising Quillaja Saponaria saponins or a fish feed comprising Quillaja Saponaria saponins as part of their daily diet. It may further be preferred that the Quillaja Saponaria saponins, the composition comprising Quillaja Saponaria saponins or the fish feed comprising Quillaja Saponaria saponins is supplied to fish for at least 7 days. As mentioned previously the feed may be administered in the freshwater stage (as a prevention) or in the seawater/saltwater phase (as a prevention and/or treatment) - such administration may be for at least 7 days in order to obtain a sufficient preventive effect or treatment.

It may further be preferred that the Quillaja Saponaria saponins, the composition comprising Quillaja Saponaria saponins or the fish feed comprising Quillaja Saponaria saponins is administered orally. The sea lice life cycle consists of 10 different stages (plus the egg stage), with a moult between each one. Thus, in a preferred embodiment it may be preferred that the Quillaja Saponaria saponins, the composition comprising Quillaja

Saponaria saponins or the fish feed comprising Quillaja Saponaria saponins is effective on Chalimus I, Chalimus II, Chalimus III, Chalimus IV, pre-adult, adult sea lice stages and gravid females infested or re-infested on fish.

Thus, one aspect of the present invention pertains to the use of Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins for the manufacture of a medicament for the treatment and/or prevention of ectoparasitic copepod infection or infestation in fish.

Another aspect of the present invention pertains to a method of treating and/or preventing ectoparasitic copepod infection or infestation in fish, said method comprising oral administering to said fish Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins.

The use or the prevention or the treatment according to the present invention may be a reduction of the total number of Chalimus I, Chalimus II, Chalimus III, Chalimus IV and adult sea lice stages infested or re-infested on fish compared to a population of fish fed an approved fish feed comprising no Quillaja Saponaria saponins.

The use or the prevention or the treatment may be combined with other traditional fish-specific antiparasitic compounds or other chemical, medical or natural agents against ectoparasites. Such strategies include but is not limited to oral treatments such us emamectin benzoate and diflubenzuron. Chemical or medical agents include but is not limited to immersion treatments such as hydrogen peroxide, deltamethrin, cypermethrin and azametiphos.

Method for the manufacture of a fish feed

There are numerous recipes for making fish feed . When combined with approved fish feed ingredients Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins may be used as a dry powder or solubilized in liquids including but not limited to oil before applied to the feed (e.g. pellet).

Including Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins in fish feed includes using state of the art technology well known for the person skilled in the art.

Thus, in a further aspect the present invention pertains to methods for the manufacture of compositions (such as fish feed) as defined above. In general these methods include steps of provising a composition comprising Quillaja

Saponaria saponins together with any of the fish feed ingredients defined above.

In an embodiment Quillaja Saponaria saponins may be mixed together with the fish feed ingredients prior to formation of the fish feed pellets.

Thus, in an even further embodiment the invention provides a method for manufacturing a fish feed, comprising the steps of:

i) combining Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins with approved fish feed ingredients, and (ii) obtaining a fish feed comprising Quillaja Saponaria saponins.

In another embodiment the present invention pertains to the manufacture of a fish feed, comprising the steps of:

i) combining Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins with approved fish feed ingredients, ii) forming fish feed and (iii) obtaining a fish feed comprising Quillaja Saponaria saponins.

The fish feed may be formulated as a pressed fish feed, pelleted fish feed, expanded fish feed, extruded fish feed and wet semimoist feed .

The combining of Quillaja Saponaria saponins or a composition comprising Quillaja Saponaria saponins with approved fish feed ingredients may be performed by use of mixing the ingredients in a feed mixer prior to further processing of the feed into fish feed . It goes without saying that the fish feed obtained may comprise the ingredients, constituents etc. stated under the heading "administration and formulation".

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples and figure, wherein

Examples

Example 1

Toxicity of Quillaja saponaria saponins on Caligus.

The experiment was carried out under in vitro conditions. Ten adults Caligus rogercresseyi in total, five females plus five males, were deposited in a Petri plaque and incubated with 0, 0.5, 50 and 500 ppm of Quillaja high extract, and maintained in an incubator chamber at 12° C + 0.5 and under controlled oxygen saturation. Survival of Caligus was evaluated 24 hours after challenge. Each challenge condition was assessed in duplicate.

The Quillaja high extract comprises 20 % w/w Quillaja Saponaria saponins.

Survival of Caligus was evaluated 24 hours after challenge. Each challenge condition was assessed in duplicate.

As can be seen from Figure 1, it was observed that mortality of Caligus presented a dose response pattern achieving a 50, 85 and 100% of mortality at 0.5, 50 and 500 ppm of Quillaja high extract (which thus corresponds to 0.1, 10 and 100 ppm of Quillaja Saponaria saponins) respectively. Both females and males presented a similar sensitivity to each dose of Quillaja Saponaria saponins tested. Example 2

Effect of Quillaja saponaria saponins on the infection rate with Caligus in Atlantic salmon (Salmo salar).

The experiment was carried out at Fundacion Chile aquaculture research facilities (Puerto Montt, Chile). 1250 Atlantic salmon smolt of 120 g which had no previous contact with Caligus were distributed in 25 fiberglass tanks of 0.4 m ³ with full salinity water and acclimated during 21 days fed with a commercial diet (i.e.

control diet with the commercial name "CPK 4mm"). Later fish from this group were fed to satiation with : (i) control (diet 1) or (ii) 1000 ppm of Quillaja high extract (which corresponds to 200 ppm Quillaja saponaria saponins (diet 2) during 42 days. Fish were challenged by immersion with Caligus rogercresseyi (42 copepodids per fish) by adding the said copepodids to the water in the tanks where the fish were maintained, with the objective to evaluate the effect of each diet on growth fish were weighted at the start, and at day 52 of the trial. Uneaten feed was collected in all tanks after finishing the feeding, weighted and stored . Each experimental condition was carried out in five replicates.

As can be seen from Figure 2, feeding with diet 2 resulted in a 44.3% of reduction of number of Caligus per fish compared with the group fish fed diet 1. From figures 3A-3C it is clear that there are no statistically significant differences on SGR (specific growth rate); SFR (specific feed rate) and FCR (feed conversion rate) between both groups: diet 1 = control diet and diet 2 = 1000 ppm of Quillaja high extract (which corresponds to 200 ppm Quillaja saponaria saponins). Example 3

Atlantic salmon maintained in a productive seawater site were distributed in two units each one conformed by 12 cages, unit 1 and unit 2, establishing two groups with similar characteristics. Each of these units where fed with either a commercial standard diet (Control) or a test diet which included Quillaja saponaria saponins. At the start of the trial the average body weight of fish was 2.5 kg in both groups. The recording of the number of Caligus was done in five cages per unit, selected according to their location : two index cages and three central cages. The counting of Caligus was carried out at the start of the feeding regimen and 33 days after to a continuous feeding regimen .

Considering the average values of the infestation rate of each group, in figure 5 it is shown that consistently with the results obtained with the in vivo trial (Example 2), fish from Unit 2, fed with the Test Diet, presented a 43.6% of reduction of the infestation rate with Caligus compared with fish from Unit 1 (fed with Control Diet) 127% and 225% of infestation rate, respectively. By way of exemplification these percentages are calculated as follows : the difference between the

infestation rate of both groups, expressed in percentage is (127/225)* 100 = 56.44%. 100-56.44=43.56 or 43.6% less Caligus in the group fed with the diet added with Quillaja saponaria saponins. 127% and 225% are the infestation rates expressed in percentage, presented in figure 5.

The Infestation rate was calculated as follows : (Number of Caligus at day 33)- (Number of Caligus at basal time) = X. Then the infestation rate will be

X/(Number of Caligus at basal time) . Then the infestation rate expressed as percentage is : X/(Number of Caligus at basal time)* 100.

Even more, similar to the results of the in vivo trial (Example 2), the effect exerted by Test Diet on the infestation rate with Caligus, was attributable to the reduction of all stages of Chalimus. The productive parameters SGR, SFR and FCR were not affected by any of the treatments.

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