Title: Prevention and treatment of HSMI

Field of invention

The present invention relates to the use of a compound for the preparation of a nutritional and/or pharmaceutical composition for the prevention and treatment of heart and skeletal muscle inflammation (HSMI). More specifically, the present invention relates to the reduced HSMI mortality, and to an improved growth during periods with HSMI challenges.

Background of invention

Heart and skeletal muscle inflammation (HSMI) was first reported from salmon farmers along the Norwegian mid-coast in 1999, and this area still reports the highest number of outbreaks in Norway, even though the disease is now found along the entire coast. Today, the incidence of HSMI in Norway is increasing. In 2006 at least 94 locations were affected by the disease, compared to 83 incidences in 2005 and 54 in 2004 (National Veterinary Institute in Norway, 2006). Severe and repeated outbreaks of HSMI result in high economical losses due to lowered appetite and high mortality, and even though mortality and duration of an outbreak can be variable, the morbidity may be very high.

The disease was originally described in salmon 5-9 months post sea transfer in Norway. Outbreaks now seem to occur during the whole year, but most usual during spring (May-June) and autumn (October-November).

Affected fish may become anorexic and display abnormal swimming, but may also be slow moving without any other external signs of disease. HSMI is reported to cause histopathological lesions in heart and skeletal muscle. The lesions associated with HSMI is partly similar to those described for two other serious diseases in modern salmon farming; pancreas disease (PD) and cardiomyopathy syndrome (CMS), making it difficult to distinguish between them. The main difference between HSMI and PD is the absence of pancreatic lesions in HSMI. The causes and metabolic mechanisms behind these diseases are not yet solved.

Today, no vaccines or therapeutic treatment for avoiding or reducing the extent of HSMI outbreaks in practical salmon farming exist, and as far as we know, the present study is the first nutritional study describing any effects on HSMI mortality.

It is thus an object of the present invention to provide a compound that can prevent or treat HSMI, or reduce the effect of the HSMI mortality.

Further, it is an object of the present invention to provide a compound that can increasing the body weight of fish that experiences HSMI.

A first aspect of the present invention thus relates to the use of a compound represented by;

(a) the general formula R"-COO-(CH2)2n+i-X-R\ wherein X is a sulphur atom, a selenium atom, an oxygen atom, a SO group or a SO ₂ group; wherein n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an

oxygen atom, a SO group and a SO ₂ group; and R" is a hydrogen atom or an alkyl group containing from 1 to 4 carbon atoms;

or

(b) the general formula (I),

wherein R1 , R2, and R3 represent i) a hydrogen atom; or ii) a group having the formula CO-R in which R is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, and the main chain of said R contains from 1 to 25 carbon atoms; or iii) a group having the formula CO-(CH ₂ WrX-R', wherein X is a sulphur atom, a selenium atom, an oxygen atom, a SO group or a SO ₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH ₂ group, a SO group and a SO ₂ group; iv) an entity selected from the group comprising -PO ₃ CH ₂ CHNH ₃ COOH (serine), PO ₃ CH ₂ CH ₂ NH ₃ (ethanolamine), P0 ₃ CH ₂ CH ₂ N(CH ₃ ) ₃ (choline), PO ₃ CH ₂ CHOHCH ₂ OH (glycerol) and PO ₃ (CHOH) ₆ (inositol); wherein R1 , R2, and R3 are chosen independently from i), ii), iii), or iv), but at least one of R1 , R2, or R3 is defined by iii);

or

(c) the general formula (II),

wherein A1 , A2 and A3 are chosen independently and represent an oxygen atom, a sulphur atom or an N-R4 group in which R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms; wherein R1 , R2, and R3 represent i) a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 23 carbon atoms; or ii) a group having the formula CO-R in which R is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, and the main chain of said R contains from 1 to 25 carbon atoms; or iii) a group having the formula CO-(CH ₂ WrX-R', wherein X is a sulphur atom, a selenium atom, an oxygen atom, a SO group or a SO ₂ group; n is an integer of 0 to 1 1 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH ₂ group, a SO group and a SO ₂ group;

iv) an entity selected from the group comprising -PO ₃ CH ₂ CHNH ₃ COOH (serine), PO ₃ CH ₂ CH ₂ NH ₃ (ethanolamine), PO ₃ CH ₂ CH ₂ N(CHs) ₃ (choline), PO ₃ CH ₂ CHOHCH ₂ OH (glycerol) and PO ₃ (CHOH) ₆ (inositol); wherein R1 , R2, and R3 are chosen independently from i), ii), iii), or iv), but at least one of R1 , R2, or R3 is defined by iii);

or

a salt, prodrug or complex of the compounds according to (a)-(c),

for the manufacturing of a nutritional or pharmaceutical composition, for the prevention or treatment of heart and skeletal muscle inflammation (HSMI) of fish, and/or for reducing the mortality of fish with HSMI or which are susceptible for HSMI.

A preferred embodiement relates to the use of the compounds defined in claim 1 for improving the cardiosomatic index (CSI) of fish with HSMI or which are susceptible for HSMI.

A further aspect of the invention relates to a compound represented by;

(a) the general formula R"-COO-(CH ₂ ) _2n +i-X-R\ wherein X is a sulphur atom, a selenium atom, an oxygen atom, a SO group or a SO ₂ group; wherein n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a SO group and a SO ₂ group; and R" is a hydrogen atom or an alkyl group containing from 1 to 4 carbon atoms;

or

(b) the general formula (I),

wherein R1 , R2, and R3 represent i) a hydrogen atom; or ii) a group having the formula CO-R in which R is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, and the main chain of said R contains from 1 to 25 carbon atoms; or iii) a group having the formula CO-(CH ₂ WrX-R', wherein X is a sulphur atom, a selenium atom, an oxygen atom, a SO group or a SO ₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH ₂ group, a SO group and a SO ₂ group; iv) an entity selected from the group comprising -PO ₃ CH ₂ CHNH ₃ COOH (serine), PO ₃ CH ₂ CH ₂ NH ₃ (ethanolamine), P0 ₃ CH ₂ CH ₂ N(CH ₃ ) ₃ (choline), PO ₃ CH ₂ CHOHCH ₂ OH (glycerol) and PO ₃ (CHOH) ₆ (inositol); wherein R1 , R2, and R3 are chosen independently from i), ii), iii), or iv), but at least one of R1 , R2, or R3 is defined by iii);

or

(c) the general formula (II),

wherein A1 , A2 and A3 are chosen independently and represent an oxygen atom, a sulphur atom or an N-R4 group in which R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms; wherein R1 , R2, and R3 represent i) a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 23 carbon atoms; or ii) a group having the formula CO-R in which R is a linear or branched alky! group, saturated or unsaturated, optionally substituted, and the main chain of said R contains from 1 to 25 carbon atoms; or iii) a group having the formula CO-(CH ₂ WrX-R', wherein X is a sulphur atom, a selenium atom, an oxygen atom, a SO group or a SO ₂ group; n is an integer of 0 to 11 ; and R' is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the main chain of said R' contains from 13 to 23 carbon atoms and optionally one or more heterogroups selected from the group comprising an oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a CH2 group, a SO group and a SO ₂ group; iv) an entity selected from the group comprising -PO ₃ CH ₂ CHNH ₃ COOH (serine), PO ₃ CH ₂ CH ₂ NH ₃ (ethanolamine), PO ₃ CH ₂ CH ₂ N(CH ₃ ) _S (choline), PO ₃ CH ₂ CHOHCH ₂ OH (glycerol) and PO ₃ (CHOH) ₆ (inositol); wherein R1 , R2, and R3 are chosen independently from i), ii), iii), or iv), but at least one of R1 , R2, or R3 is defined by iii);

or

a salt, prodrug or complex of the compounds according to (a)-(c),

for the manufacturing of a nutritional or pharmaceutical composition, for increasing the body weight of fish with HSMI or which are susceptible for HSMI.

Further embodiments for the aspects claimed in the claim 1 to 3, are defined in the subclaims 4 to 19.

In such an embodiment the composition is administered orally.

In a further embodiment the composition is fed to fish, such as tilapia, Atlantic cod, Atlantic halibut, European sea bass and salmonides, preferable Atlantic salmon or rainbow trout.

Preferable, the composition is administered or fed to the fish for a period of 12 weeks, preferable 6 weeks, more preferable 3 weeks or less.

Preferred compounds of the present invention are non β-oxidizable fatty acid such as tetradecylthioacetic acid (TTA), tetradecylselenoacetic acid and 3-Thia-15- heptadecyne.

Further preferred compounds are modified phospholipids or mono-, di- or tri- acylglycerides.

In a preferred embodiment the composition comprises an amount of a compound defined by (a)-(c) in the range of about 0.05% to 1 ,25%, related to the weight of the feed, or more preferable 0.1 % to 0.50%, related to the weight of the feed, and more preferable about 0,25%, related to the weight of the feed.

Another preferred embodiment relates to a composition comprising an amount of a compound defined by an amount of a compound defined by (a)-(c) in the range of

about 0.05% to 0,5%, related to the weight of the total biomass, or more preferable 0.1 % to 0.25%, related to the weight of the total biomass.

A further aspect of the invention relates to a fish feed containing the compounds defined in claim 1 for the prevention or treatment of heart and skeletal muscle inflammation (HSMI) of fish, and/or for reducing the mortality of fish with HSMI or which are susceptible for HSMI, and/or for increasing the body weight of fish with HSMI or which are susceptible for HSMI.

A preferred embodiment of said fish feed comprises an amount of a compound defined by an amount of a compound defined by (a)-(c) in the range of about 0.05% to 0,5%, related to the weight of the total biomass, or more preferable 0.1% to 0.25%, related to the weight of the total biomass, such a about 0,25%, or more preferable about 0,50%, related to the weight of the total biomass.

Another preferred embodiment of said fish feed comprises an amount of a compound defined by (a)-(c) in the range of about 0.05% to 1 ,25%, related to the weight of the feed, or more preferable 0.1 % to 0.50%, related to the weight of the feed, or more preferable about 0,25%, and more preferable about 0,50% of the weight of the feed.

Experimental section

The experimental research is conducted by using tetradecylthioacetic acid (TTA) as the representative compound, but it must be emphasized that the present invention is not restricted to this specific compound, but is defined by the compounds stated in the claims. Further, the inventors have used Atlantic salmon as the test organisms, but it is anticipated that the present compounds also will have an effect on other fishes which are subjected to the same disease.

The compounds of the present invention are blocked in various positions and cannot be beta-oxidized. Without being bound by theory, we believe that this

inhibited β-oxidation will lead to an accumulation of fatty acids in the cell, and this accumulated amount will function as a positive feedback mechanism to increase the β-oxidation of normal fatty acids.

Figure legends

Figure 1 shows the cumulative mortality during a natural outbreak of HSMI from end of May until 20 ^th July 2007 for farmed 0+ Atlantic salmon given four different diets, either a low-fat control diet (C1 ), high-fat control diet (C2), C2 added 0.25% TTA (E1 ) or E1 added 0.2% Carnitine (E2).

Figure 2 shows the total mortality during a natural HSMI outbreak in spring for the control group or the experimental group of farmed 0+ Atlantic salmon. Significant differences between dietary groups are indicated by different letters on the bars.

Figure 3 shows the changes in thermal growth coefficient (TGC) and feed conversion ratio (FCR) for 0+ salmon fed the control diets or experimental diets, throughout the experimental period.

This present study which forms the basis for the present invention was performed to evaluate the effect of the compounds of the present as dietary supplements on fish that were challenged with an HSMI outbreak. We have surprisingly shown that the compounds of the present invention reduces the mortality of HSMI challenged fish, and also that the compounds have an effect on the growth capability during such a period.

Materials and methods

Fish and treatments

The present study was conducted in seawater at AKVAFORSK research station at the Norwegian west coast (Averøy), using Atlantic salmon (0+) transferred to sea in November 2006. In mid-March 2007 the fish were stocked into eight net pens

(5x5x5 m) with 550 fish in each. A randomized block design was used as the experimental setup, with all the net pens located on the same pier at the research facilities. Initial mean body weight was 221 ±1 gram, resulting in a weight of 1585±62 grams at the final sampling in September. Four different diets were fed during the first six weeks (period 1 ). The four different diets were: a commercial basic diet (C1-CPK 200), C1 coated with 3% fat (2% rapeseed oil and 1 % capelin oil) (C2), C2 added 0.25% TTA (E1 ) and E1 added 0.2% carnitine (E2). The experimental diets were prepared by coating the basic diet in a blender. Carnitine was dissolved in distilled water and coated onto the surface of the feed pellets for E2, to a final inclusion of 0.2%. The similar amount of water was coated onto the two other high-dietary fat diets (C2 and E1 ) and all three diets were dried at room temperature. C2 was then coated with 2% rapeseed oil and E1 and E2 were coated with TTA dissolved in the same amount of rapeseed oil, to make a final dietary level of 0.25%. Finally, 1 % of capelin oil was coated onto the three diets. From week seven onwards (period 2), the fish were fed the same diet as in feeding period 1 , but without added TTA. In period 2, the commercial basic diet was adjusted to a bigger fish size in accordance to the manufacturer's guidelines and the pellet size was increased (CPK 500). The two basic diets were produced by Biomar AS (Norway) (Table 1 ). All the diets were fed in excess in duplicated net pens, and feed waste sampling was performed during both feeding periods.

Table 1 Chemical content of feed used during the experimental period

Basic diet period 1 Basic diet period 2

(6 weeks) (app. 20 weeks)

Dry matter (DM) (%) 93.2 94.5

Protein (% of DM) 45.9 42.5

Lipid (% of DM) 31.8 29.3

Ash (% of DM) 9.1 8.2

NFE ¹ (%) 6.4 14.5

¹ NFE = Nitrogen free extracts

Sampling and analyses

All diets were analysed gravimetrically for dry matter (DM) after drying at 105 ⁰ C (16-18 h), and ash (flame combustion followed by 3-4 h at 550 ⁰ C). The content of crude proteins was calculated as N*6.25 using the semi-micro-Kjeldahl method (Kjeltec-Auto System, Tecator, Hόganas, Sweden), and crude lipid after diethyl ether extraction in a Soxtec analyser (Tecator, Hόganas, Sweden) after HCI-hydrolysis (Stoldt, 1952). Proportion of nitrogen free extract (NFE) was estimated as: NFE = DM - (fat + protein + ash).

Three times ten fish were randomly sampled at start of the experiment. Thereafter ten fish from each cage, representing the average weight, were sampled every third week until August (2007). Individual body weights (to the nearest gram) and heart weights (to the nearest 0.1 mg) were registered for sampled fish. All sampled fish were anesthetized (MS 222 metacaine, 0.1 g L ^"1 ) and killed by a blow to the head.

During the disease outbreak dead fish were recorded daily. The local veterinary service (Nordvest Fiskehelse AS, Kristiansund, Norway) was contacted immediately when mortality increased and a disease outbreak was suspected. The veterinarians dissected organs from 11 fish from five of the eight pens, representing all dietary treatments. In addition, two fish from the cage where the fish originated from, were analysed. Samples were diagnosed by the National Veterinary Institute of Norway (NVI, Oslo) using histological analysis. Myocarditis was observed in all fish, but since pancreas was not included in the two first fish groups submitted, a final diagnosis of HSMI was only given in the last group submitted. The diagnosis was then confirmed on all diets except the one added only TTA (EI ).

Every sixth week blood samples were taken from the caudal vein using EDTA vacuum tubes, from nine fish per cage. Plasma was obtained by centrifugation at

4 ⁰ C and 2000 rpm for 10 minutes. Equal volumes of plasma from three fish were pooled in each of three cryotubes, frozen in liquid nitrogen and then stored at -8O ⁰ C for later analyses.

Calculations

Weight gain was calculated as thermal growth coeffiscient (TGC) (c.f. Cho, 1992): TGC = (W _b ^1/3 - W _a ^1/3 ) x (∑T)-\ where W _b is the final weight, W ₃ is the initial weight and ∑T is the sum of day degrees during the period. In order to simplify the figures, the TGC is multiplied by 1000. Feed conversion ratio (FCR) was calculated as (kg feed fed) x (kg final biomass-kg initial biomass + kg dead fish) ^"1 . Cardiosomatic index (CSI) was calculated as heart weight (g) x (body weight (g)) ^'1 x 100. Relative percent survival (RPS) was calculated as described by Ahmend (1981 ) 100% x (1 - (% mortality in the experimental group x (% mortality in the control group) ^"1 )).

Statistical analyses

If not otherwise is stated, all statistical analyses were performed using the SAS software package (SAS Institute Inc. 1990). Data were analysed using analyses of variance (ANOVA). The model used mortality, CSI, FCR, TGC or urea concentration in plasma as dependent variable, and block, side of the pier, sampling point and dietary treatment as class variables. Experimental units were net pens. Proportion of the total variation explained by the model is expressed by R ² and calculated as the marginal contribution of the mean square of the parameter (type III sum of squares). Significant differences among variables within treatments were indicated by least-square means (Ismeans) comparison. The level of significance is chosen at p<0.05, and the results are presented as Ismeans ± s.e.m. (standard error of the mean), if not otherwise is stated.

Results

In late May mortality began to increase, and HSMI was confirmed to be the cause of death. In the beginning of June the mortality declined, indicating the disease outbreak to be over. Cumulative mortality is shown in figure 1 for the four diets

used. Since no differences were observed between the two control diets or between the experimental diets added TTA, the diets were pooled into one control and one TTA group. Hence, no effect of different dietary levels of fat or supplementation of Carnitine was observed.

Both the side of the pier and diet was found to significantly influence total mortality, together explaining 81 % of the observed variation in the model. Supplementing diets with TTA was found to significantly reduce total mortality during the HSMI outbreak, as shown in figure 2. The results show an RPS between the TTA-group and the control diets of 45%.

Body weight at the end of the experimental period was influenced by both block (p<0.001 ) and diet (p=0.04). TTA diets resulted in a higher final body weight (1606 ± 29 grams) than control diets (1580 ± 31 grams). For both control and TTA-fed fish thermal growth coefficient (TGC) was lowest and feed conversion ratio (FCR) was highest between week six and twelve after experimental start. During this period fish fed TTA supplemented diets had significantly (p=0.04) higher TGC, and seemingly improved feed conversion (p= 0.23) (figure 3). Except for this period, which coincides with the disease outbreak, no differences in growth between the two groups were observed.

Due to individual variations in the cardiosomatic index, we found it most correct to use the mean value of the three samplings performed after the outbreak (in June and July). Both diet (p=0.05), block (p=0.008) and side of the pier (0.04) were found to significantly influence CSI, together explaining 91 % of the observed variation. CSI was significantly higher in fish fed a TTA-supplemented diet (0.137 ± 0.004) compared to control fish (0.132 ± 0.003).

Discussion

The timing of the natural outbreak of HSMI, in May and June, is in accordance to Kongtorp et al. (2006) and Watanabe et al. (2006). Kongtorp et al. (2006) studied a commercial farm with a history of recurrent outbreaks of HSMI, and sampled the

first fish to be diagnosed with HSMI during May followed by a clinical outbreak of HSMI in June. The presented outbreak of HSMI also coincides in time with outbreaks in several commercial farms along the Norwegian coast, and the National Veterinary Institute in Norway (2006) reports May-June and October- November to be the periods with highest number of samples to be diagnosed.

The mortality started at different times in the net pens, and the mortality was highest at one side of the pier. This may indicate that the disease was spread among cages and also that the cages experienced different infectious pressure during the outbreak. A block design was used in our experiment, making us able to observe dietary effects even though the mortality was higher at some positions. Our study was not designed to differentiate and measure infectious pressure, but the results support the suggestions made that HSMI is contagious and spread in a cage to cage pattern (Kongtorp et al., 2004a, b).

Myocarditis was observed in all sampled fish submitted to NVI during the disease outbreak. This lesion is present in fish affected by HSMI, but also reported for other diseases such as PD. These three diseases together cause significant losses for the salmon farming industry, and so far no good methods for avoiding and combating these diseases are available. The vaccine companies work hard to develop vaccines with good effect on the viruses, but still no vaccines are available in the market. For PD some nutritional studies have been performed, showing an effect of additional antioxidants as vitamin E on mortality rates. The present study is, however, the first to show any dietary effect on mortality caused by HSMI.

The heart index was found to be higher in fish fed the TTA-supplemented diets compared to the control.

During the disease period, fish fed TTA had a higher growth rate and a lower feed conversion ratio, which may be due to the better health situation in these groups. This period during spring is suggested to be a critical period for salmon transferred

to sea as underyearlings, measured as lowered muscle fat, condition factor and growth performance.