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Title:
FEED COMPOSITION
Document Type and Number:
WIPO Patent Application WO/2011/031166
Kind Code:
A2
Abstract:
A feed composition is described which is suitable for feeding of fish in order to prevent or reduce the symptoms related to Heart and Skeletal Muscle Inflammation (HSMI) disease in the fish.

Inventors:
VECINO JOSE LUIS GONZALEZ (NO)
WADSWORTH SIMON (NO)
NORDRUM SIGVE (NO)
Application Number:
PCT/NO2010/000336
Publication Date:
March 17, 2011
Filing Date:
September 14, 2010
Export Citation:
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Assignee:
EWOS INNOVATION AS (NO)
CHEMOFORMA LTD (CH)
AKER BIOMARINE ASA (NO)
VECINO JOSE LUIS GONZALEZ (NO)
WADSWORTH SIMON (NO)
NORDRUM SIGVE (NO)
International Classes:
A23K1/16; A23K1/18
Domestic Patent References:
WO2007056823A12007-05-24
WO2008136739A12008-11-13
WO2004098311A12004-11-18
WO2005107493A12005-11-17
WO2009058799A12009-05-07
WO2009108067A22009-09-03
WO2008051091A12008-05-02
WO2010027788A12010-03-11
WO2007061314A22007-05-31
WO2009131467A12009-10-29
WO2008097103A12008-08-14
Foreign References:
EP0322114A21989-06-28
Other References:
KONGTORP ET AL.: "Heart and skeletal muscle inflammation in Atlantic salmon, Salmo salar L.: a new infectious disease", JOURNAL OF FISH DISEASE, vol. 27, no. 6, 2004, pages 351 - 358, XP008053704, DOI: doi:10.1111/j.1365-2761.2004.00549.x
ELIASSEN ET AL.: "Isolation of heart and skeletal muscleinflammation virus (HSMIV) from Salmon", THE 6TH INTERNATIONAL SYMPOSIUM ON VIRUSES OF LOWER VERTEBRATES, 2004
"R Development Core Team", 2009, R FOUNDATION FOR STATISTICAL COMPUTING, article "R: A Language and Environment for Statistical Computing"
LUNN, D.J.; THOMAS, A.; BEST, N.; SPIEGELHALTER, D.: "WinBUGS - a Bayesian modelling framework: concepts, structure, and extensibility", STATISTICS AND COMPUTING, vol. 10, 2000, pages 325 - 337
GELMAN, A.; RUBIN, D.B.: "Inference from iterative simulation using multiple sequences, Statistical", SCIENCE, vol. 7, 1992, pages 457 - 511
Attorney, Agent or Firm:
ACAPO AS (Bergen, NO)
Download PDF:
Claims:
CLAIMS

1. A feed composition for fish for the prevention and/or treatment of Heart and skeletal muscle inflammation disease comprising conventional feed ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, characterized in that more than 20 % of the total fatty acids are n-3 fatty acids.

2. A feed composition according to claim 1 , wherein preferably more than 23 % of the total fatty acids are n-3 fatty acids. 3. A feed composition according to claim 1 , wherein preferably more than 24 % of the total fatty acids are n-3 fatty acids.

4. A feed composition according to any of the previous claims wherein the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than about 7 % of the total fatty acids. 5. A feed composition according to claim 4, wherein the content of

eicosapentaenoic acid 20:5 n-3 (EPA) is more than about 8 % of the total fatty acids, more preferably than 9 %, most preferably more than about 10%, such as about 10.2%.

6. A feed composition according to any of the previous claims wherein the

protein:lipid ratio (w/w) in the feed is higher than 2, preferably higher than 2.7.

7. A feed composition according to claim 6, wherein the proteins account for more than 50 % (by weight) and the lipids account for less than 20 % (by weight) of the feed composition.

8. A feed composition according to claim 7, wherein the proteins account for more than 52 % (by weight) and the lipids account for about 8 % (by weight) of the feed composition.

9. A feed composition according to any of claims 1 or 2 characterized in that the composition is for salmonids and that the composition has a reduced content of lipids and an increased content of proteins when compared to commercial feed compositions used for salmonids at the same age, size and/or developmental stage. 0. A feed composition comprising according to any of the previous claim, wherein the content of n-6 fatty acids are less than 1 1 % of the total fatty acids, preferably in the range of 7-11 %, most preferably about 10%.

11.A feed composition according to claim 10, wherein the content of n-3 fatty acids are more than 23% and the level of n-6 fatty acids are less than 11 % of the total fatty acids.

12. A feed composition according to any of the previous claim, the ration of n-3 to n-6 ratio fatty acids is higher than 1.6, preferably more than 2.2, most preferably between 2.3 and 3.4. 13. A feed composition according to any of the previous claims wherein a portion of said lipids is South American Fish oil.

14. A feed composition according to any of the previous claims wherein the content of added vegetable oil is less than 0.5 %, preferably 0.2 % by weight of the composition, and that the oil is preferably added as a hydrolysate. 15. A feed composition according to any of the previous claims wherein the

composition comprises hydrolysed phospholipids, preferably about 0.1-0.2 % by weight of the diet, most preferably more than 0.2 % by weight of the diet.

16. A feed composition according to any of the previous claims wherein the

composition comprises krill meal, preferably about 5 % by weight, most preferably more than 5 % by weight of the diet.

17. A feed composition according any of the preceding claims, wherein the feed further comprises nucleotides and/or manna-oligosaccharides and/or antiinflammatory compounds such as flavonoids.

18. A feed composition according to any of the previous claims, wherein the proteins account for more than 50 % (by weight) of the composition, wherein the lipids account for less than 20 % (by weight), wherein more than 20 % of the total fatty acids are n-3 fatty acids. 19. A feed composition according to claim 18, wherein the proteins account for more than 52 % (by weight) of the composition, and wherein the lipids account for less than 18 % (by weight), preferably for 18 % of the diet, wherein more than 23 % of the total fatty acids are n-3 fatty acids.

20. A feed composition according to any of the previous claims characterized in that the composition is substantially as indicated for the anti-HSMI diet 1 or for the anti HSMI diet 2 in table 1 or table 2.

21. Use of the feed composition according to any of the previous claims 1-20 for the manufacturing of a pharmaceutical composition and/or nutritional composition and/or functional feed for the prophylaxis and/or treatment of Heart and skeletal muscle inflammation disease.

22. Use of the feed composition according to claim 20, to prevent or reduce the

symptoms related to Heart and skeletal muscle inflammation disease in fish.

23. Use according to claim 20, wherein the fish is a salmonid, flat fish or any other fish species suitable for aquaculture, preferably the species Atlantic salmon {Salmo salar).

24. Use according to claim 20, for effective recovery following a Heart and skeletal muscle inflammation infection and/or reduction of the risk of re-infection.

25. A method for feeding of fish which are susceptible to a Heart and skeletal muscle inflammation infection characterized in that a composition according to any of the claims 1 -20 is fed to the fish in the period previous to the challenge by an infection, during the infection or after the fish has been infected.

26. A method according to claim 25 characterized in that the composition is fed for a period of at least 8 weeks pre infection, more preferably of 10-16 weeks, most preferably of 16-20 weeks and 8-20 weeks post infection.

27. A feed composition for fish comprising conventional feed ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, characterized in that more than 20 % of the total fatty acids are n-3 fatty acids.

28. A feed composition according to claim 27, wherein preferably more than 23 % of the total fatty acids are n-3 fatty acids.

29. A feed composition according to claim 28, wherein preferably more than 24 % of the total fatty acids are n-3 fatty acids.

30. A feed composition according to any of the claim 27-03 wherein the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than about 7 % of the total fatty acids.

31.A feed composition according to claim 30, wherein the content of

eicosapentaenoic acid 20:5 n-3 (EPA) is more than about 8 % of the total fatty acids, more preferably than 9 %, most preferably more than about 10%, such as about 10.2%. 32. A feed composition according to any of the claims 27-31 wherein the protein:lipid ratio (w/w) in the feed is higher than 2, preferably higher than 2.7.

33. A feed composition according to claim 32, wherein the proteins account for more than 50 % (by weight) and the lipids account for less than 20 % (by weight) of the feed composition. 34. A feed composition according to claim 33, wherein the proteins account for more than 52 % (by weight) and the lipids account for about 18 % (by weight) of the feed composition.

35. A feed composition comprising according to any of the claims 27-34, wherein the content of n-6 fatty acids are less than 11 % of the total fatty acids, preferably in the range of 7- %, most preferably about 10%.

36. A feed composition according to claim 35, wherein the content of n-3 fatty acids are more than 23% and the level of n-6 fatty acids are less than 1 % of the total fatty acids.

37. A feed composition according to any of the claims 27-36, wherein the ration of n- 3 to n-6 ratio fatty acids is higher than 1.6, preferably more than 2.2, most preferably between 2.3 and 3.4.

38. A feed composition according to any of the claims 27-37, wherein a portion of said lipids is South American Fish oil.

39. A feed composition according to any of the claims 27-38, wherein the content of added vegetable oil is less than 0.5 %, preferably 0.2 % by weight of the composition, and that the oil is preferably added as a hydrolysate.

40. A feed composition according to any of the claims 27-39, wherein the

composition comprises hydrolysed phospholipids, preferably about 0.1-0.2 % by weight of the diet, most preferably more than 0.2 % by weight of the diet.

41.A feed composition according to any of the claims 27-40, wherein the

composition comprises krill meal, preferably about 5 % by weight, most preferably more than 5 % by weight of the diet. 42. A feed composition according any of the claims 27-41 , wherein the feed further comprises nucleotides and/or manna-oligosaccharides and/or anti-inflammatory compounds such as flavonoids.

43. A feed composition according to any of the claims 27-42, wherein the proteins account for more than 50 % (by weight) of the composition, wherein the lipids account for less than 20 % (by weight), wherein more than 20 % of the total fatty acids are n-3 fatty acids.

44. A feed composition according to claim 43, wherein the proteins account for more than 52 % (by weight) of the composition, and wherein the lipids account for less than 18 % (by weight), preferably for 8 % of the diet, wherein more than 23 % of the total fatty acids are n-3 fatty acids.

45. A feed composition according to any of previous claims 27-44, characterized in that the composition is substantially as indicated for the anti-HSMI diet 1 or for the anti HSMI diet 2 in table 1 or table 2.

46. A feed composition for fish comprising conventional feed ingredients such as proteins, lipids, vitamins, carbohydrates and minerals characterized in that the protein:lipid ratio (w/w) in the feed is higher than 2, preferably higher than 2.7 to prevent or reduce the symptoms related to Heart and skeletal muscle

inflammation disease in the fish.

47. A feed composition according to claim 46, wherein the protein:lipid ratio (w/w) in the feed is higher than 2.7

48. A feed composition according to claim 46, characterized in that the comprised proteins account for more than 50 % (by weight) of the diet, preferably more than 52 % and the comprised lipids account for less than 20 % (by weight), preferably for 18 % of the diet.

49. A feed composition according to claim 46, wherein more than 20 % of the total fatty acids are n-3 fatty acids, preferably more than 23 %, most preferably 24%.

50. A feed composition according to claim 46, wherein the composition comprises more than 23% n-3 fatty acids and less than 11 % n-6 FA of the total fatty acids.

51.A feed composition according to claim 46 or 49, wherein the content of

eicosapentaenoic acid 20:5n-3 is more than 7 % of the total fatty acids, preferably more than 9 %, most preferably 10.2%.

Description:
FEED COMPOSITION

FIELD OF THE INVENTION

The present invention relates to a feed composition for fish comprising conventional feed ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, a use of the feed composition as well as to a method for feeding of fish which are susceptible to a Heart and skeletal muscle inflammation infection.

BACKGROUND TO THE INVENTION

Heart and skeletal muscle inflammation disease (HSMI) is a serious infectious disease of Atlantic salmon with a high potential for transmission from infected to healthy fish. Since the first occurrence in 1999, an increasing number of outbreaks of the disease have been registered. Thus, after 68 outbreaks registered in 2003 and 54 in 2004, the number has been increasing every year. In 2007 the disease was diagnosed at 162 seawater sites along the Norwegian coast and in 144 in 2008 (Figure 1 ). These outbreaks are causing huge losses to the Norwegian salmon industry since on sites that are affected, very high numbers of fish (>90%) may suffer heart lesions months after the disease event, significantly reducing their growth potential. Most outbreaks are reported in January, June, July and October, in fish transferred to seawater. The disease has a great economical impact to the

Norwegian salmon farming industry, and a single fish farming company, reported losses up to 10 million NOK per year due to the disease 5-9 months later. However, there are indications that HSMI is also relevant in other species than those belonging to Salmonids.

Clinical signs of HSMI are typically reduced appetite and aberrant swimming behaviour. There are no obvious external signs, but at autopsy the heart appears pale, soft and flaccid. HSMI produces inflammation of the heart which results in death of heart cells. These damages can already be seen in early stages of the disease and can continue for several months. Mortalities increase with an increasing degree of heart inflammation. The inflammation and cell death can be spread to other organs and tissues such as the red skeletal muscle and the liver causing additional severe damages also to these organs.

SUBSTITUTE SHEET The infectious nature of HSMI has been proven, as disease could be induced in healthy fish by injecting homogenized tissue from fish diagnosed with HSMI, and further be transferred to healthy cohabitant fish (Kongtorp et ai, 2004. Heart and skeletal muscle inflammation in Atlantic salmon, Salmo salar L.: a new infectious disease. Journal of Fish Disease: Vol. 27(6), p. 351-358). HSMI is caused by a virus, which has been isolated (Eliassen et al., 2004. Isolation of heart and skeletal muscleinflammation virus (HSMIV) from Salmon. The 6th International Symposium on Viruses of Lower Vertebrates, Poster 19). However, since the virus has not been fully characterised yet, vaccines against HSMI have not been developed so far. In addition, current challenge models of the disease at the lab only reproduce the histopathology observed in the field, not mortality. Thus, there is a clear need for alternative treatments and methods in order to reduce the negative effects of this severe infection. An object of the present invention is therefore to find new strategies and treatments which can be used to positively affect the disease development in HSMI-infected fish as well as in fish susceptible to the infection.

SUMMARY OF THE INVENTION A first aspect of the present invention relates to a feed composition for fish for the prevention and/or treatment of Heart and skeletal muscle inflammation disease comprising conventional feed ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, characterized in that more than 20 % of the total fatty acids are n-3 fatty acids.

In a preferred embodiment, more than 23 % of the total fatty acids are n-3 fatty acids, and more preferable more than 24 % of the total fatty acids are n-3 fatty acids.

In a currently preferred embodiment is the content of eicosapentaenoic acid 20:5 n-3 (EPA) more than about 7 % of the total fatty acids. More preferable, the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than about 8 % of the total fatty acids, more preferably than 9 %, most preferably more than about 10%, such as about 10.2%. A preferred embodiment contains the amounts of EPA indicated in table 1. In a preferred embodiment is the the protein:lipid ratio (w/w) in the feed higher than 2, preferably higher than 2.7. Preferable, the proteins account for more than 50 % (by weight) and the lipids account for less than 20 % (by weight) of the feed composition. More preferable, the proteins account for more than 52 % (by weight) and the lipids account for about 18 % (by weight) of the feed composition.

A preferred embodiment relates to a feed composition for salmonids wherein the composition has a reduced content of lipids and an increased content of proteins when compared to commercial feed compositions used for salmonids at the same age, size and/or developmental stage.

In a further preferred embodiment is the content of n-6 fatty acids less than 11 % of the total fatty acids, preferably in the range of 7-11 %, most preferably about 10%.

In a preferred embodiment is the ratio of n-3 to n-6 ratio fatty acids is higher than 1.6, preferably more than 2.2, most preferably between 2.3 and 3.4. In a preferred embodiment is a portion of said lipids South American Fish oil.

Preferable, the feed composition contains less than 0.5 %, preferably 0.2 % by weight of the composition of added vegetable oil.

In a preferred embodiment is the lipid (oil) added as a hydrolysate.

A preferred embodiment comprises hydrolysed phospholipids, preferably about 0.1- 0.2 % by weight of the composition, most preferably more than 0.2 % by weight of the composition. A preferred embodiment comprises krill meal, preferably about 5 % by weight, most preferably more than 5 % by weight of the diet.

A preferred embodiment comprises nucleotides and/or manna-oligosaccharides and/or anti-inflammatory compounds such as flavonoids.

A preferred embodiment relates to a feed composition, wherein the proteins account for more than 50 % (by weight) of the composition, wherein the lipids account for less than 20 % (by weight), wherein more than 20 % of the total fatty acids are n-3 fatty acids. More preferable, the proteins account for more than 52 % (by weight) of the composition, and wherein the lipids account for less than 18 % (by weight), preferably for 18 % of the diet, wherein more than 23 % of the total fatty acids are n- 3 fatty acids. A preferred embodiment relates to a feed composition substantially as indicated for the anti-HSMI diet 1 or for the anti HSMI diet 2 in table 1 or table 2. A second aspect of the present invention relates to a feed composition as indicated above for the manufacturing of a pharmaceutical composition and/or nutritional composition and/or functional feed for the prophylaxis and/or treatment of Heart and skeletal muscle inflammation disease.

A preferred embodiment relates to prevention and reducing the symptoms related to Heart and skeletal muscle inflammation disease in fish.

In a preferred embodiment is the fish a salmonid, flat fish or any other fish species suitable for aquaculture, preferably the species Atlantic salmon (Salmo salar). In a preferred embodiment is the use for effective recovery following a Heart and skeletal muscle inflammation infection and/or reduction of the risk of re-infection.

A third aspect of the present invention relates to a method for feeding of fish which are susceptible to a Heart and skeletal muscle inflammation infection characterized in that a composition as indicted above is fed to the fish in the period previous to the challenge by an infection, during the infection or after the fish has been infected.

Preferable, the composition is fed for a period of at least 8 weeks pre infection, more preferably of 10-16 weeks, most preferably of 16-20 weeks and 8-20 weeks post infection.

A fourth aspect of the present invention relates to a feed composition for fish comprising conventional feed ingredients such as proteins, lipids, vitamins, carbohydrates and minerals, characterized in that more than 20 % of the total fatty acids are n-3 fatty acids.

Preferable, more than 23 % of the total fatty acids are n-3 fatty acids.

Preferable, more than 24 % of the total fatty acids are n-3 fatty acids.

More preferable, the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than about 7 % of the total fatty acids. More preferable, the content of eicosapentaenoic acid 20:5 n-3 (EPA) is more than about 8 % of the total fatty acids, more preferably than 9 %, most preferably more than about 10%, such as about 10.2%.

Preferable, the proteinrlipid ratio (w/w) in the feed is higher than 2, preferably higher than 2.7 or the proteins account for more than 50 % (by weight) and the lipids account for less than 20 % (by weight) of the feed composition, more preferable wherein the proteins account for more than 52 % (by weight) and the lipids account for about 18 % (by weight) of the feed composition. In a preferred embodiment is the content of n-6 fatty acids are less than 11 % of the total fatty acids, preferably in the range of 7-11 %, most preferably about 10%.

In a preferred embodiment is the content of n-3 fatty acids are more than 23% and the level of n-6 fatty acids are less than 11 % of the total fatty acids.

In a preferred embodiment is the ration of n-3 to n-6 ratio fatty acids is higher than 1.6, preferably more than 2.2, most preferably between 2.3 and 3.4.

In a preferred embodiment is a portion of said lipids South American Fish oil.

Preferable, the content of added vegetable oil is less than 0.5 %, preferably 0.2 % by weight of the composition, and that the oil is preferably added as a hydrolysate.

Preferable, the composition comprises hydrolysed phospholipids, preferably about 0.1-0.2 % by weight of the diet, most preferably more than 0.2 % by weight of the diet.

Preferable, the composition comprises krill meal, preferably about 5 % by weight, most preferably more than 5 % by weight of the diet.

Preferable, the feed further comprises nucleotides and/or manna-oligosaccharides and/or anti-inflammatory compounds such as flavonoids.

Preferable, the proteins account for more than 50 % (by weight) of the composition, wherein the lipids account for less than 20 % (by weight), wherein more than 20 % of the total fatty acids are n-3 fatty acids, and more preferable the proteins account for more than 52 % (by weight) of the composition, and wherein the lipids account for less than 18 % (by weight), preferably for 18 % of the diet, wherein more than 23 % of the total fatty acids are n-3 fatty acids.

In a preferred embodiment is the composition substantially as indicated for the anti- HSMI diet 1 or for the anti HSMI diet 2 in table 1 or table 2.

A fifth aspect of the present invention relates to a feed composition for fish comprising conventional feed ingredients such as proteins, lipids, vitamins, carbohydrates and minerals characterized in that the protein:lipid ratio (w/w) in the feed is higher than 2, preferably higher than 2.7 to prevent or reduce the symptoms related to Heart and skeletal muscle inflammation disease in the fish.

Preferable, the protein:lipid ratio (w/w) in the feed is higher than 2.7 Preferable, proteins account for more than 50 % (by weight) of the diet, preferably more than 52 % and the comprised lipids account for less than 20 % (by weight), preferably for 18 % of the diet.

Preferable, more than 20 % of the total fatty acids are n-3 fatty acids, preferably more than 23 %, most preferably 24%.

Preferable, the composition comprises more than 23% n-3 fatty acids and less than 1 1 % n-6 FA of the total fatty acids. Preferable, the content of eicosapentaenoic acid 20:5n-3 is more than 7 % of the total fatty acids, preferably more than 9 %, most preferably 10.2%.

It will be appreciated that features of the invention described in the foregoing can be combined in any combination without departing from the scope of the invention.

DESCRIPTION OF THE INVENTION

Embodiments of the invention will now be described, by the way of examples with reference to the following diagrams, wherein

Figure 2 shows the modelled cumulative probabilities of myocardiac scores between feeds at 10, 12, 14 and 16 weeks. The graph gives the probability of the myocardiac score being equal or lower than the one read on the x-axis. Figure 3 shows the model coefficients (± 95% credible intervals) of the myocardiac score model comparable between feeds at 10, 12, 14 and 16 weeks post-challenge. That means that if the 95% credible interval do not overlap the red dashed line denoting the coefficient of control diet for a particular week, the difference can be considered significant at 5% level. Also, if the feeds are compared across the subplots, the weeks that do not have the 95% credible intervals crossing the zero line (not drawn) can be considered significantly different from the 10 weeks.

Figure 4 shows the modelled cumulative probabilities of the epicard scores for each feed at 10, 12, 14 and 16 weeks. The graph gives the probability of the epicardiac score being equal or lower than the one read on the x-axis. Even though differences were not significant between the different diets on epicard scores, there is a clear trend that the epicard scores are improved by feeding of anti-HSMI 1 or 2.

Figure 5 shows the model coefficients (± 95% credible intervals) of the epicardiac score model comparable between feeds at 10, 12, 14 and 16 weeks post-challenge. That means that if the 95% credible interval do not overlap the red dashed line denoting the coefficient of control diet for a particular week, the difference can be considered significant at 5% level. Also, if the control feeds are compared across the subplots, the weeks that do not have the 95% credible intervals crossing the zero line (not drawn) can be considered significantly different from the 0 weeks.

Figure 6 shows the modelled cumulative probabilities of liver score between feeds within sampling weeks. The graph gives the probability of the score being equal or lower than the one read on the x-axis.

Figure 7 shows model coefficients (± 95% credible intervals) of the liver score model comparable between feeds at 10, 12, 14 and 16 weeks post-challenge. That means that if the 95% credible interval do not overlap the red dashed line denoting the coefficient of control diet for a particular week, the difference can be considered significant at 5% level. Also, if the control feeds are compared across the subplots, the weeks that do not have the 95% credible intervals crossing the zero line (not drawn) can be considered significantly different from the 10 weeks.

Figure 8 shows the average weight gain and 95% confidence interval during the 8- week feeding period pre-challenge. Figure 9 shows the level of EPA in heart tissue of Atlantic salmon 8, 10, 12 and 16 weeks post-challenge.

EXPERIMENTAL SECTION

Experiment

The dietary effect on HSMI was tested in a feeding experiment with infected Atlantic salmon {Salmo salar). The salmon smolts used for the experiment had all been fed the same commercial diet for a period of 10 weeks during the acclimatization period. All the fish were in good condition and showed good and rapid adaptation to sea- water.

Feeding experiment:

The trial consisted of 2 phases. Duration of the first part was an 8-week period pre- challenge, which was conducted at a feeding trial facility. After this period the fish were transferred to a disease challenge facility and fish were acclimatized for 2 weeks. After this period the second part of the study comprising challenge, feeding post-challenge lasted for 16 weeks (i.e. 16 weeks post-challenge).

A total of 945 Atlantic salmon Salmo salar, pit-tagged, average start weight 110g, seawater adapted were distributed in 9 tanks (1.0 x 1.0 m., 50 cm depth; 500 litre volume) at the feeding trial facility. Three diets were randomly allocated among the 9 tanks (3 tanks per diet) and fed to the fish for a period of 8 weeks. The amount of feed offered during the 8 week period was based on measured or estimated biomass in the tanks. The daily feed rations offered varied between 0.8 - 1.1 % fish body weight per day. Trial feeds given were adjusted through the trial to compensate for fish growth. Fish were kept in seawater with ambient temperature and salinity and diurnal photoperiod, 24:0 (light (L): dark (D)). Water temperature and salinity were measured every day in a representative tank. After the 8-week period, fish were weighed and length-measured, pit-tag recorded and transferred to the disease challenge facility. Once at the challenge facility, the fish were allocated to 18 tanks (2 labs x 9 tanks, i.e. 6 tanks per diet) keeping the same dietary history as during the previous 8 weeks. Fish were left to get acclimatized to the tanks at the challenge facility for 2 weeks and then challenged against HSMI. Fish were anaesthetized prior to challenge to the HSMI virus. The HSMI virus was isolated from sick fish during a clinical outbreak of HSMI. All fish were challenged by intramuscular injection (0.1 ml on each side close to the lateral line) of virus collected from cell culture supernatant of HSMI virus. The general condition of the fish was recorded daily and any mortality was summarized on a daily basis (no mortality was expected from challenge). All other mortalities, due to any causes, occurring during the course of the experiment, were registered. Diets:

Fish were either fed a control diet or the two different experimental diets anti-HSM1 1 and anti-HSMI 2 according to the present invention. The pellet size of all diets was 3.5 mm. The diets comprised conventional feed ingredients which are commonly comprised in fish feed such as proteins, lipids, carbohydrates, vitamins, minerals. The compositions of the diets were as follows (Table 1 ):

Table 1 : Moisture, protein, lipid, ash content (all weight % of diet) as well as fatty acid (FA) composition (% of total fatty acids) and n-3/n-6 fatty acid ratio of the diets used in the feeding experiment.

Control diet anti-HSMI 1 anti-HSMI 2

Moisture 4.7 8.4 8.5

Protein** 45.1 52.7 53.4

Ash 7.7 9.2 9.4

Lipid* 31.8 18.8 18.6

Myristic acid C14 3.1 4.9 7.2

Pentadecanoic acid C15 0.3 0.4 0.5

Palmitic acid C16 14.1 14.9 19.3

16:1 n-7 4.1 6.1 7.3

Palmitoleic acid C16:1 n-9 <0.1 <0.1 <0.1

Hexadecadienoic acid 16:2 0.4 0.3 0.4

Hexadecatrienoic acid 16:3 0.3 0.3 0.4

Hexadecatetranoic acid 16:4 0.2 1.3 0.5

Stearin acid C18 2.3 2.8 2.3

C18:1 n-7 2.5 3.3 4.3

Oleic acid C18:1 n-9 34.9 23.5 17.2

Linoleic acid C18:2n-6 11.2 8.9 5.7 alfa-Linolenic acid C18:3n-3 4.8 3.1 1.7 gamma-Linolenic acid C18:3n-6 0.1 0.2 0.2

Stearidonic acid C18:4n-3 1.2 1.5 1.9

Arachidic acid C20 0.4 0.3 0.2

Gadolein acid C20:1 <0.1 <0.1 <0.1

20:1 n-7 0.1 0.3 0.3

20:1 n-9 3 3.5 4

Eicosadienoic acid C20:2n-6 0.2 0.2 0.2

C20:3 n-3 0.1 <0.1 0.1

C20:3 n-6 <0.1 0.1 0.1

C20:4 n-3 0.4 0.5 0.5

C20:4 n-6 0.4 0.6 0.6

EPA C20:5 n-3 5 10.5 9.8

Behenic acids C22 0.3 0.1 0.1

22:1 n-11 2.6 3.1 3.7

22:1 n-9 0.4 0.5 0.7

C22:4n-6 <0.1 0.1 0.1

C22:5n-3 0.6 1.2 0.8

C22:5n-6 0.1 0.2 0.1

DHA C22:6n-3 6 7.3 9.1

Lignoceric acid C24 <0.1 <0.1 <0.1

Tetracosenic acid C24:1 0.7 0.6 0.9

Total monounsaturated FA (%) 48.4 40.8 38.4

Total polyunsaturated FA (%) 31.1 35.9 32.1

Total saturated FA (%) 20.5 23.3 29.5 sum n-3-FA (% of FA) 18.1 23.9 23.9 sum n-6-FA (% of FA) 12 10.2 7

Total (%) 100 100 100 ratio n-3 FA /n-6 FA 1.5 2.3 3.4 "lipids were determined according to the method of Folch, proteins were estimated from elemental N (applied factor 6.25).

Table 2: Lipid classes (%) in the diets fed during the feeding experiment.

Sampling:

Fish were sampled in regular intervals after 8, 10, 12, 14 and 16 weeks and histological samples of heart muscle and liver were analyzed in respect to histopathological changes as described below. A total of 360 fish were analyzed from the feeding experiment.

Further treatment and analysis of the samples:

The histopathology of heart and liver were conducted by professional veterinary services. Samples were fixed in 10% neutral phosphate buffered formalin. Formalin fixed samples were prepared for histological examination by standard paraffin wax techniques and stained with haematoxylin and eosin (H&E).

Analyzed parameters:

1. Heart inflammation:

The histological changes have been evaluated on the basis of a predetermined categorization of changes in the heart. The focus was on the cardiac inflammation:

- Endothelial changes in the borderline between the compact layer and the spongious layer of the heart were important parameters in the characterization. At early stages swelling and profileration was important, followed by infiltration with mononuclear inflammatory cells

- Infiltration of inflammatory cells in the compact layer, focal or diffuse in appearance

- Infiltration of inflammatory cells in the spongious layer, focal or diffuse in appearance accompanied by degeneration and necrosis of myocytes, and swelling of endothelial cells lining the spongious myofibres.

Scores were available from 8, 10, 12, 14 and 16 weeks post challenge. Scores from 8 weeks were omitted from the analysis since only a total of 5 fish were examined (2 for one diet and 3 for another). Histological changes in epicard and myocard were ranked according to a non-continuous score grade from 0 to 4 (0 indicates no pathology, normal tissue; 4 intense inflammation). A description of each of the scores for epicard and myocard are included in Table 3.

Table 3: Inflammation scores for the epicard and myocard of HSMI infected Atlantic salmon.

Pathological description - epicard Pathological description - myocard

Score 0: No pathological changes Score 0: No pathological changes observed. observed.

Score 1 : Focal / multifocal (2-4 foci) of Score 1 : Vascular changes in the small inflammatory cells lifting the epicardial vessels of the compact layer characterized layer from the surface of the heart, by enlarged endothelial cells, typically typically 2-3 cell layers thick. Limited stretching out. No involvement of compact number (countable) of mononuclear layer extending into the compact layer of inflammatory cells infiltrating the the ventricle.

epicardium.

If there is only involvement of epicard

with minor or very little compact layer

involvement; max 1.5 score (diffuse

and >5 cell layer thick for most of the

inflamed area).

Score 2: Diffuse infiltration of Score 2: Focal to multifocal inflammatory inflammatory cells (mononuclear) >5 foci (2-5 foci) of the compact layer and/or in cell layers thick in most of the epicard the spongious part (2-5 foci). Extension present. The infiltration of cells is typically seen along small vessels and multifocal to diffuse and can involve perivascular infiltration,

parts of or the entire epicardium

available for assessment.

Score 3: Diffuse infiltration of Score 3: The changes in the compact layer inflammatory cells (mononuclear) >10 are multifocal or diffuse in areas and cell layers thick in most of the epicard typically concentrate along small blood present. Moderate pathological vessels. Combined with focal or multifocal changes consisting of high number changes in the spongious layer.

(uncountable) of inflammatory cells in

the epicardium

Score 4: Diffusely thickened (>15 cell Score 4: Widespread to diffuse infiltration layers) epicard in more than ¾ of the of inflammatory cells in the compact layer layer present. Severe pathological and involving the spongious layer in a changes characterized by intense multifocal pattern. Degeneration and or infiltration of inflammatory cells in the necrosis of muscle fibers may be/are seen, epicardium, Atrium can also be involved with

inflammatory changes 2. Liver inflammation:

Liver histopathology was studied by changes related to micro- and macrovesicular steatosis. This is characterized by varying degrees of vacuole-formation in the cytoplasm of the hepatocytes. Score grading followed a non-continuous score from 0 to 5, being 5 the score indicating the highest degree of steatosis of the liver (fatty liver). A description of each of the grading scores is as follows.

- Score 0: individual formation of vacuoles in the cytoplasm, involving less than 10% of the hepatocytes and including less than 25% of the area of the individual hepatocytes.

- Score 1: individual formation of vacuoles in the cytoplasm, involving less than 25% of the hepatocytes and including less than 25% of the area of the individual hepatocytes.

- Score 2:individual formation of vacuoles in the cytoplasm, involving less than 50% of the hepatocytes and including less than 50% of the area of the individual hepatocytes.

- Score 3: individual formation of vacuoles in the cytoplasm, involving less than 75% of the hepatocytes and including less than 75% of the area of the individual hepatocytes.

- Score 4: individual formation of vacuoles in the cytoplasm, involving less than 90% of the hepatocytes and including less than 80% of the area of the individual hepatocytes.

- Score 5: individual formation of vacuoles in the cytoplasm, involving more than 90% of the hepatocytes and including more than 80% of the area of the individual hepatocytes.

Statistical analysis:

All data preparation and simulation output analysis was conducted with the R language (R: A Language and Environment for Statistical Computing, R

Development Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2009, ISBN 3-900051-07-0, http://www.R-project.org). Individual weight gain data was analysed by using a linear mixed-effects model (multilevel model) fitted between the response and predictor by allowing the intercepts to vary from tank to tank to account for the tank level correlations and variability. The model was a mixed-effects linear model estimated with the Imer function in the Ime4 package. The treatment estimates were based on posterior simulation (n=2500) with 95% credible intervals as absolute and proportional to the reference level (control diet).

Histopathological scores were analysed by using a multilevel ordered categorical logistic regression because the data are multinomial. The model was written in BUGS language (Lunn, D.J., Thomas, A., Best, N., and Spiegelhalter, D. (2000) WinBUGS - a Bayesian modelling framework: concepts, structure, and extensibility. Statistics and Computing, 10:325-337) and fitted with JAGS (JAGS=Just Another Gibbs Sampler, Plummer, M. (2009) JAGS Version 1 .03 manual, http://www- fis.iarc.fr/~martyn/software/jags/). Vague non-informative uniform priors [0,100] were given for the variance parameters, and vague non-informative normal priors N(0, 1.0E+4) for all other parameters. 25000 "burn-in" simulation runs were used to adapt the Markov Chain Monte Carlo (MCMC) before subsequent 2500 runs that were used for inference. Three chains were run in parallel, i.e. there were a total of 7500 simulations for inference. These were thinned so that only every 10th simulation was saved to reduce the size of saved objects and to reduce the effects of

autocorrelation. In effect, the posterior density is based on 750 draws from the posterior probability distribution. Convergence of the MCMC simulation was judged by the so-called Gelman-Rubin convergence diagnostic (Gelman, A. and Rubin, D.B. (1992) Inference from iterative simulation using multiple sequences, Statistical Science, 7, 457-51 1 ). Results and conclusion:

The results from the feeding experiment show that it is possible to reduce the impact of HSMI disease through the diet. By using specific diet formulations it is possible to reduce both heart damage and liver steatosis during disease outbreak The main changes observed in the heart between the dietary groups were in the myocardium, while no clear differences between diets were observed in the epi- cardium. Regarding the myocardium, at 10 weeks post-challenge no significant differences in the myocardiac scores between diets could be observed since the 95% credible intervals crossed the doted line (Figure 3a). As the disease progressed the scores seemed to get worse for the control feed at 14 and 16 weeks in comparison to 10 and 12 weeks (negative coefficients for control feed in figure 3). At 2 weeks post- challenge the myocardiac started to show a trend towards differences between diets and 99% of the fish fed diet anti-HSM1 1 had myocardiac scores equal or lower than 1 , and for diets control and anti-HSMI 2 around 96% of the fish had scores equal or lower than 1 (Figure 2b); The statistical analysis showed that diet anti-HSM1 had higher coefficients resulting in better (lower) scores than the control feed at week 2 however at this time it can be considered only trend since the 95% credible intervals crossed lightly the doted line (Figure 3b). At week 14 post-challenge, 62% of fish fed control diet had myocardiac scores equal or lower than 1 , compared to 86% with diet anti-HSMI 2 and 96% with diet anti-HSMI 1 (Figure 2c). At week 16 post-challenge, 94% of fish fed diet anti-HSMI 1 had myocardiac scores equal or lower than 1 compared to 85% and 51 % for diets anti-HSMI 2 and control respectively (Figure 2d). Differences between diets anti-HSMI 1 and control at 14 and 16 weeks were statistically significant since higher coefficients resulting in significantly better (lower) myocardiac scores were observed with feed anti-HSMI 1 compared to the control feed (Figure 3c). Similar positive effects but much milder and less certain were observed with feed anti-HSMI 2, however not statistically significant (Figure 3d).

Regarding epicardium, at 10 weeks post-challenge for all feeds 91-92% of the epicardiac scores are equal or lower than , and about 40% of the scores were zero for all the feeds (Figure 4a). At 12 weeks post-challenge the feed anti-HSMI 2 had slightly lower probability (81 %) of fish having epicardiac scores equal or lower than 1 compared to 90% for diet control and 92% for diet anti-HSMI 1 , although these differences were not significant (Figure 4b). At week 14 post-challenge, there was a trend for feed anti-HSMI 1 with a higher probability of 85% of having epicardiac scores equal or lower than 1 compared to 69% and 63% probability for the diets anti- HSMI 2 and control, respectively (Figure 4c). At week 16 post-challenge, diets anti- HSMI 1 and anti-HSMI 2 appeared to have higher probabilities (90% and 85% respectively) of epicardiac scores equal or lower to 1 than the control feed (74%). Although this cannot be claimed significant, a clear trend can be seen for feed anti- HSMI 1 to improve the pathological status of the fish (Figure 4d). Thus, clear effects of the dietary treatments on the inflammation status of the fish were observed from 12 weeks onwards (Figure 3-4), where especially the diet anti- HSMI 1 resulted in a reduced inflammation of the heart as indicated by higher probability of having low histopathological scores compared to the control diet. Even more prominent differences were observed later on in the feeding experiment after 4 and 16 weeks of feeding the anti-HSMI diets 1 and 2 when compared to the control group. This means that fish fed the anti-HSMI diet 1 had significantly less damage compared to the control diet. The anti-HSMI diet 2 also showed reduction in heart damage but not as good as HSMI diet 1. The results surprisingly but clearly show the potential of these dietary compositions in the modulation and treatment of the HSMI disease in Atlantic salmon.

Fish suffering from HSMI disease have been described to have changes in the liver. Thus, livers are yellowish, showing an increased fat content (commonly known and hepatic steatosis) and sometimes necrotic foci are observed where cells are vacuolated and pyknotic. Feeding of the new dietary compositions to HSMI infected Atlantic salmon surprisingly also affected the pathological status of the liver (Figures 6-7). At 10 weeks post-challenge the fish fed diets anti-HSMI 1 and anti-HSMI 2 had clearly higher probabilities of having low scores (i.e. score≤ 1 )(79% and 72% respectively) compared to 16% for the control diet fish (Figure 6a). The coefficients for the feeds anti-HSMI 1 and anti-HSMI 2 were significantly better (lower scores- less steatosis) at 10 weeks (figure 6a). At 14 weeks post-challenge feed anti-HSMI 1 also had higher probabilities (74%) of having low scores (i.e. indicating low degree of hepatic steatosis) compared to 40% for feed anti-HSMI 2 and to 23% for the control feed. These differences were significant lower for diet anti-HSM1 1 , while a trend was seen for diet anti-HSMI 2 when compared to the control (Figure 7c). Thus, clear effects of the anti-HSMI diets on the liver status of the fish were observed at 10 and 14 weeks (Figure 6-7), where especially the diet anti-HSMI 1 resulted in a reduced hepatic steatosis as indicated by higher probability of having low histopathological scores compared to the control diet. These effects were particularly observed at 10 and 14 weeks post-challenge.

In addition, clear significant differences in weight gain were observed between diets pre-challenge (Figure 8). Fish grew 89.82 g, 109.90 g and 107.28g respectively for diets control, anti-HSM1 1 and anti-HSMI 2 (i.e. 23% and 20% more than the control diet when compared with the anti-HSMI diets) after the 8-week feeding period before challenge. The exact mechanism(s) behind the surprising effect of reducing heart inflammation in HSMI-infected fish by the new dietary compositions according the present invention are not clear yet. A high content of n-3 fatty acids, especially of the fatty acid eicosapentaenoic acid (EPA 20:5n-3) is beneficial. Further, a reduced content of lipids combined with an increased content of protein, when compared to

conventional feed compositions used for growth of the same species, seems to be beneficial for the modulation of the HMSI disease.

Figure 9 shows that the level of the fatty acid eicosapentaenoic acid (EPA 20:5n-3) accumulates in the heart tissue, and without being bound by any mechanism it is believed that this increased level of EPA at the right place in the fish body, i.e. in the heart tissue, is favorable for the HSMI-infected fish.

Furthermore, in a preferred embodiment according to the present invention South American fish oil which contains approximately double EPA than North Atlantic fish oil, is used to increase the content of EPA in the anti-HSMI diets.

Furthermore, a reduction of the content of n-6 fatty acids, typically found in vegetable oils, seems to be beneficial in the modulation of the HSMI disease. The results also indicate that a reduced n-3/n-6 ratio such as being in the range of 2.3-3.4 can be beneficial.

The liver of HSMI infected fish is affected by the disease as described above. This can have an impact on lipid digestibility in the fish. In a preferred embodiment of the present invention vegetable oils are added to the diets in a hydrolysed form. It also seems to be an advantage to include hydrolysed phospholipids to the diet. It can also be beneficial to add krill meal and/or krill oil to the diet. Preferably, the diet may further comprise nucleotides, anti-inflammatory compounds and/or manna- oligosaccharides (MOS). It is assumed that the feed composition according to the present invention is also relevant for other teleost species than salmonids which can be affected by HSMI.

It will be appreciated that the features of the invention described in the foregoing can be modified without departing from the scope of the invention.

Definitions of terms and abbreviations:

"FA" is the abbreviation used for "fatty acids" "n-3 fatty acids" are a family of unsaturated fatty acids that have in common a final carbon-carbon double bond in the n-3 position, which is the third bond from the methyl end of the fatty acid. These fatty acids are also known as omega 3 fatty acids. "n-6 fatty acids" are a family of unsaturated fatty acids that have in common a final carbon-carbon double bond in the n-6 position, which is the sixth bond from the methyl end of the fatty acid. These fatty acids are also known as omega 6 fatty acids.

By the term "n-3/n-6 ratio" is meant the ratio between fatty acids belonging to the n-3 family and those belonging to the n-6 family in the composition or diet.

"Protein/lipid ratio" means the ratio between proteins and lipids comprised in the composition or diet (each of them related to the weight of the diet).

"HSMI" is the abbreviation used for "Heart and skeletal muscle inflammation disease" The term "conventional feed ingredients" refers to ingredients which are commonly in fish feeds such as proteins, lipids, carbohydrates, vitamins, minerals, etc. The ingredients can be derived from marine, vegetable, animal by-products and/ or any other relevant sources and can be used in any suitable combination.

The term "nucleotides" refer to molecules comprising a nitrogenous base (purine or pyrimidine base), a five-carbon sugar and one to three phosphate groups.