Method for treatment or prevention of gill disease

Abstract

Composition for treatment of mucus on fish gills for a therapeutic or a prophylactic treatment of an amoebic gill disease in fish, where the composition comprises an extruded fish feed supplemented with arginine; said fish feed comprising protein, binder, fat, vitamins and minerals; and a total arginine content of the fish feed is at least 3.0% (wt/wt) of a total feed weight.

Claims

1. A method of increasing a mucus viscosity of a fish in need thereof, comprising determining that the fish is in need of increased mucus viscosity, and providing an extruded fish feed to the fish in need thereof, wherein the extruded fish feed comprises a total arginine content of at least 3.0% (wt/wt) of total feed weight.

2. The method of claim 1, wherein the extruded fish feed comprises protein, binder, fat, vitamins, and minerals.

3. The method of claim 1, wherein the fish comprises a salmonid fish.

4. The method of claim 3, wherein the salmonid fish comprises at least one of rainbow trout or Atlantic salmon.

5. A method of increasing a polysaccharide concentration in the mucus of a fish in need thereof, comprising determining that the fish is in need of increased polysaccharide concentration in the mucus, and providing an extruded fish feed to the fish in need thereof, wherein the extruded fish feed comprises a total arginine content of at least 3.0% (wt/wt) of total feed weight.

6. The method of claim 5, wherein the extruded fish feed comprises protein, binder, fat, vitamins, and minerals.

7. The method of claim 5, wherein the fish comprises a salmonid fish.

8. The method of claim 7, wherein the salmonid fish comprises at least rainbow trout or Atlantic salmon.

9. A method of increasing a lysozyme concentration in the mucus of a fish in need thereof, comprising determining that the fish is in need of increased lysozyme concentration in the mucus, and providing an extruded fish feed to the fish in need thereof, wherein the extruded fish feed comprises a total arginine content of at least 3.0% (wt/wt) of total feed weight.

10. The method of claim 9, wherein the extruded fish feed comprises protein, binder, fat, vitamins, and minerals.

11. The method of claim 9, wherein the fish comprises a salmonid fish.

12. The method of claim 11, wherein the salmonid fish comprises at least rainbow trout or Atlantic salmon.

Description

(1) In the following are described examples of preferred embodiments.

(2) FIGS. 1-3 show survival in Atlantic salmon following challenge with the amoeba Paramoeba perurans in different studies;

(3) FIG. 4 shows in vitro survival of Paramoeba pemaquidensis after 72 hours of incubation with fish mucus;

(4) FIG. 5 shows viscosity of Atlantic salmon mucus;

(5) FIG. 6 shows concentration of lysozyme in Atlantic salmon mucus;

(6) FIG. 7 shows concentration of polysaccharides in Atlantic salmon mucus;

(7) FIG. 8 shows concentration of lysozyme in Atlantic salmon mucus;

(8) FIG. 9 shows in vitro survival of Paramoeba pemaquidensis after 48 hours of incubation with fish mucus; and

(9) FIG. 10 shows in vitro survival of Paramoeba perurans after 48 hours of incubation with fish mucus.

EXAMPLE 1

(10) The test was carried out with Atlantic salmon (S. salar) for 65 days in 250 l tanks containing salt water at 35 ppt salinity and at a water temperature of 16° C. There were 30 fish per tank with an average weight of 121 g at the start of the test and two tanks per diet.

(11) The fish were acclimated and fed a control diet for five weeks prior to onset of the 65 day study period, then fed either the control diet or a test diet until trial end. The control diet, also termed control feed, Control 2, comprised wheat, wheat gluten, North Atlantic fish meal, soy protein concentrate, rapeseed oil, North Atlantic fish oil, astaxanthin, vitamins and minerals. The control diet was produced by extrusion cooking and was composed of 26.5% fat, 50.1% protein and 5.7% water and is representative of a commercial fish feed. The test diet, also termed test feed, Control 2+A, had the same composition as the Control 2 feed, but with arginine added at 1.0%. Arginine was added as a dry powder in the meal mix before cooking extrusion. The calculated total level of arginine in the Control 2 feed was 2.61% on an as is basis.

(12) P. perurans were harvested from Atlantic salmon held in an infection tank following the methods described in Morrison R N, Crosbie P B B, Nowak B F. 2004. (The induction of laboratory-based amoebic gill disease revisited. J. Fish Dis, 27, 445-449). After four weeks of feeding the experimental diets, the fish were challenged with a total dose of 500 cells per litre of P. perurans over a series of days (0, 8, 9, 10, 12 and 16 post infection). For the challenge water circulation was stopped in all tanks and amoeba added to each tank using a watering can containing an additional 7 l of seawater to ensure even distribution of amoebae in the tank. Water flow was reinstated after 1.5-2 h.

(13) The trial ended when the control group reached 60% mortality. The average fish weight at trial termination was 192 g. The presence of P. perurans in a selection of mortalities was confirmed by qPCR and histology.

(14) As shown in FIG. 1, fish fed the test diet Control 2+A had a 19% relative percent survival compared to fish fed the control feed. Relative percent survival is calculated as: (1−(% mortality/% control mortality))×100.

(15) Table 1 shows that the test diet was effective at reducing mortalities attributed to AGD compared to fish fed the control diet.

(16) TABLE-US-00001 TABLE 1 Summary of mortalities at 35 days post infection Diet Survival (%) Mortality (%) Control 2 37 63 Control 2 + A 49 51

EXAMPLE 2

(17) The test was carried out with Atlantic salmon (S. salar) for 144 days in 250 l tanks containing salt water at 35 ppt salinity and at a temperature of 16° C. There were 30 fish per tank with an average weight of 171 g at the start of the test and three tanks per diet.

(18) The fish were acclimated and fed a control diet for four weeks, then fed either the control diet or test diet until trial end The control diet, also termed control feed, Control 1, comprised wheat, wheat gluten, sunflower meal, North Atlantic fish meal, soy protein concentrate, faba beans, rapeseed oil, North Atlantic fish oil, astaxanthin, vitamins and minerals. The control diet was produced by extrusion cooking and was composed of 24.2% fat, 49.9% protein 5.3% ash and 6.3% water and is representative of a commercial fish feed. The test diet, also termed test feed, Control 1+A, had the same composition as the Control 2 feed, but with arginine added at 0.58%. Arginine was added as a dry powder in the meal mix before cooking extrusion. Analysis showed that the Control 1 feed contained 2.92% arginine on an as is basis whereas the Control 1+A feed for the test group contained 3.24% arginine on an as is basis.

(19) P. perurans were harvested from Atlantic salmon held in an infection tank following the methods described by Morrison et al. After four weeks of feeding the experimental diets, the fish were challenged with a total dose of 500 cells per litre of P. perurans over two days. Due to the low number of mortalities that were observed over the course of the challenge, an additional dose of amoebae (50 P. perurans cells/l) were also added on day 55 post challenge. For the challenge water circulation was stopped in all tanks and amoeba added to each tank using a watering can containing an additional 7 l of seawater to ensure even distribution of amoebae in the tank. Water flow was reinstated after 1.5-2 h.

(20) The trial ended when the control group reached 40% mortality. The average fish weight at trial termination was 391 g. The presence of P. perurans in a selection of mortalities was confirmed by qPCR and histology.

(21) As shown in FIG. 2, fish fed the test diet, Control 1+A, had a 19% relative survival compared to fish fed the Control 1 feed.

(22) Table 2 shows that the test diet was effective at reducing mortalities attributed to AGD compared to fish fed the control 1 diet.

(23) TABLE-US-00002 TABLE 2 Summary of mortalities at 74 days post infection Diet Survival (%) Mortality (%) Control 1 59.1 40.9 Control 1 + A 66.7 33.3

EXAMPLE 3

(24) The test was carried out with Atlantic salmon (S. salar) for 144 days in 250 l tanks containing salt water at 35 ppt salinity and at a temperature of 16° C. There were 30 fish per tank with an average weight of 179 g at the start of the test and three tanks per diet.

(25) The fish were acclimated and fed a control diet for four weeks, then fed either the control diet or a test diet until trial end. The control diet, also termed control feed, Control 2′, comprised wheat, wheat gluten, sunflower meal, North Atlantic fish meal, soy protein concentrate, faba beans, rapeseed oil, North Atlantic fish oil, astaxanthin, vitamins and minerals. The control diet was produced by extrusion cooking and was composed of 24.3% fat, 47.7% protein, 5.6% ash and 7.1% water and is representative of a commercial fish feed. The test diet, also termed test feed, Control 2′+A′, had the same composition as the Control 2′ feed, but with arginine added at 0.58%. Arginine was added as a dry powder in the meal mix before cooking extrusion. Analysis showed that the Control 2′ feed contained 2.75% arginine on an as is basis whereas the Control 2′+A′ feed for the test group contained 3.30% arginine on an as is basis.

(26) P. perurans were harvested from Atlantic salmon held in an infection tank following the methods described by Morrison et al. After four weeks of feeding the experimental diets, the fish were challenged with a total dose of 500 cells per litre of P. perurans over two days. Due to the low number of mortalities that were observed over the course of the challenge, an additional dose of amoebae (50 P. perurans cells/l) were also added on day 55 post challenge. For the challenge water circulation was stopped in all tanks and amoeba added to each tank using a watering can containing an additional 7 l of seawater to ensure even distribution of amoebae in the tank. Water flow was reinstated after 1.5-2 h.

(27) The trial ended when the control group reached 40% mortality. The average fish weight at trial termination was 422 g. The presence of P. perurans in a selection of mortalities was confirmed by qPCR and histology.

(28) As shown in FIG. 3, fish fed the Control 2′+A′ feed had a 36% relative survival compared to fish fed the Control 2′ feed. Fish on the Control 2′+A′ feed had a significantly increased survival compared to fish fed the Control 2 diet at the 0.1% level of significance (Log rank, Mantel-Cox, P=0.09).

(29) Table 3 shows that the test diet was effective at reducing mortalities attributed to AGD compared to fish fed the Control 2′ diet.

(30) TABLE-US-00003 TABLE 3 Summary of mortalities at 74 days post infection Diet Survival (%) Mortality (%) Control 2′ 62.7 37.3 Control 2′ + A′ 76.1 23.9

EXAMPLE 4

(31) The test was carried out with Atlantic salmon (S. salar) for 37 days in tanks one meter in diameter containing salt water at 32.9-34.0 ppt salinity. Water temperature was varying from 11.8 to 12.1° C. There were 40 fish per tank with an average weight of 132 g at the start of the test and three tanks per diet.

(32) The control diet, also termed control feed, Control 1′, comprised wheat, wheat gluten, sunflower meal, Scandinavian fish meal, soy protein concentrate, rapeseed oil, North Atlantic fish oil, astaxanthin, vitamins and minerals. The control diet was produced by extrusion cooking and was composed of 23.2% fat, 48.0% protein, 11.1% ash and 4.9% water and is representative of a commercial fish feed. The test diet, also termed test feed, Control 1′+A′, had the same composition as the Control 1′ feed. Batches of 12.5 kg Control 1′ feed was top coated with 1% arginine for 90 seconds in a commercial bread mixer before 0.05% Nordic fish oil was added and mixing continued for another 30 seconds.

(33) At the end of the trial the fish weighed 156 g.

(34) Protocol for Culturing Mucus Samples from Fish

(35) Mucus Sampling:

(36) Skin mucus was collected individually by placing each fish on a plastic bag, gently wrapping the bag around the fish and sliding the fish out of the bag. The mucus was immediately snap frozen in liquid nitrogen and stored at minus 80° C. until analysis. Skin mucus was taken instead of gill mucus because it was not possible to collect sufficient volume of gill mucus on individual fish for viscosity, lysozyme and polysaccharide analysis. Literature discloses that the skin and gill mucus are similar in characteristics for the analysed properties and changes in skin mucus reflect changes in gill mucus.

(37) Mucus Preparation:

(38) All mucus samples are thawed and used only once, re-use after refreezing is avoided as the activity of the substances or immunological components in the mucus may be influenced by freeze-thaw cycles. Depending on the viscosity of the mucus sample, the mucus sample is used as is. If the mucus sample is very viscous, the mucus sample is spin briefly for 1 min at 1000 g to settle the cells. The resulting supernatant is used for testing.

(39) Incubation with Amoeba:

(40) All mucus samples are diluted 1:1 with cultured amoebae of the species Paramoeba pemaquidensis. Amoebae are observed and checked for survival after 4-5 hours, 24 hours, 48 hours and after 6-9 days. A stronger effect in mucus is often observed after several days of exposure.

(41) Vitality Staining:

(42) The amoeba is stained by the fluorescent dyes propidium iodine, reddead cells, and fluorescein diacetate, green-live cells, for vitality staining, following a protocol by Yokoyama et al. (Journal of Fish Diseases 1997, 20 (4), 281-286) with a modified incubation time of only 5 minutes. As an alternative, the amoeba is stained by neutral red, which stains lysosomes in live cells (Chazotte, 2010, Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, N.Y., USA). Counts are performed in triplicate for 100 cells per concentration or per individual fish mucus sample.

(43) Amoeba survival was decreased from 96 to 92 percent with 72 hours of incubation in mucus harvested from fish fed Control 1′+A′ feed as shown in FIG. 4.

EXAMPLE 5

(44) The test was carried out with Atlantic salmon (S. salar) for 34 days in tanks one meter in diameter containing salt water at 34.1-34.2 ppt salinity. Water temperature varied from 11.5 to 11.8° C. There were 20 fish per tank with an average weight of 379 g at the start of the test and one tank per diet.

(45) The control diet, also termed control feed, Control 1″, comprised wheat, wheat gluten, sunflower meal, North Atlantic fish meal, soy protein concentrate, faba beans, rapeseed oil, North Atlantic fish oil, astaxanthin, vitamins and minerals. The control diet was produced by extrusion cooking and was composed of 24.2% fat, 49.9% protein and 6.3% water and 5.3% ash and is representative of a commercial fish feed. The test diet, also termed test feed, Control 1″+A″, had the same composition as the Control 1″ feed, but with arginine added at 0.58%. Arginine was added as a dry powder in the meal mix before cooking extrusion. The analysed total level of arginine in the Control 1″ feed was 2.92% on an as is basis and in the test diet Control 1″+A″ it was 3.24% on an as is basis.

(46) At the end of the trial the fish weighed on average 470.5 g. Skin mucus was collected individually by placing each fish on a plastic bag, gently wrapping the bag around the fish and sliding the fish out of the bag. The mucus was immediately snap frozen in liquid nitrogen and stored at minus 80° C. until analysis. Skin mucus was taken instead of gill mucus because it was not possible to collect sufficient volume of gill mucus on individual fish for viscosity, lysozyme and polysaccharide analysis. Literature discloses that the skin and gill mucus are similar in characteristics for the analysed properties and changes in skin mucus reflect changes in gill mucus.

(47) Viscosity of the mucus was analysed on a Brookfield cone and plate DV3T rheometer. Mucus was centrifuged at 4000 rpm for four minutes and the viscosity of 0.5 ml of the clear particle free mucus was measured at 80 rpm at 12° C.

(48) Lysozyme activity was measured on a Varioskan Flash plate reader. 250 μl of a suspension of Micrococcus lysodeikticus in 0.4 M sodium phosphate buffer at pH 5.8 was added to 5 μl of clear particle free mucus and the absorbance was followed for 30 minutes. A decrease in absorbance at 0.001 per minute was taken as a unit of lysozyme activity.

(49) The amount of polysaccharide was measured on a Varioskan Flash plate reader. 25 μl of clear particle free mucus was mixed together with 60 μl of 2.5% phenol in water and 150 μl concentrated sulphuric acid, then incubated for 20 minutes at 100° C. After cooling to room temperature, the absorbance was measured and the concentration calculated based on standards containing glucose.

(50) FIG. 5 shows the viscosity of mucus at 80 revolutions per minute (rpm). The mucus was significantly thicker from fish in the test group fed Control 1″+A″ feed than from fish in the control group fed Control 1″ feed (P<0.0001, unpaired t test).

(51) Composition of the mucus from fish in the test group fed Control 1″+A″ feed was significantly different from those in the control group fed Control 1″ feed. The concentration of lysozyme in the mucus was significantly higher in the test group fed Control 1″+A″ feed than in the control group fed Control 1″ feed (P=0.0005, unpaired t test) as shown in FIG. 6. The concentration of polysaccharides was significantly higher in the test group fed Control 1″+A″ feed (FIG. 7).

EXAMPLE 6

(52) The test was carried out with Atlantic salmon (S. salar) for 41 days in tanks one meter in diameter containing salt water at 33.6-34.6 ppt. Water temperature ranged from 11.9° C. to 12.3° C. There were 30 fish per tank with an average weight of 322 g at the start of the test and two tanks per diet.

(53) The control diet, also termed control feed, Control 2″, comprised wheat, wheat gluten, North Atlantic fish meal, soy protein concentrate, faba beans, rapeseed oil, North Atlantic fish oil, sunflower meal, astaxanthin, vitamins and minerals. The control diet was produced by extrusion cooking and was composed of 25.8% fat, 45.0% protein, 7.3% water and 5.7% ash and is representative of a commercial fish feed. The test diet, also termed test feed, Control 2″+A″, had the same composition as the Control 2″ feed, but with arginine added at 0.86% as a dry powder in the meal mix before extrusion. The analysed total level of arginine in the Control 2″ feed was 2.63% on an as is basis, in the test diet Control 2″+A″ it was 3.14% on an as is basis.

(54) At the end of the trial the fish weighed 545 g. Skin mucus was collected individually as described in example 4.

(55) Lysozyme activity in the sampled mucus was measured as described in example 4. FIG. 8 shows the concentration of lysozyme in the mucus was higher in the test group fed Control 2″+A″ than in the control group fed Control 2″.

(56) Incubation With Amoeba:

(57) All mucus samples are diluted 1:1 with cultured amoebae either of the species Paramoeba pemaquidensis or of the species Paramoeba perurans. Amoebae are observed and checked for survival after 4-5 hours, 24 hours, 48 hours and 72 hours.

(58) Vitality staining was performed as described in example 4. P. pemaquidensis survival was significantly decreased from 97.8 to 96.8 percent (P=0.011, unpaired t test) after 48 hours of incubation and P. perurans survival was significantly decreased from 95.9 to 91.2 percent (P=0.004, unpaired t test) after 48 hours of incubation in mucus collected from fish fed Control 2″+A″ feed as shown in FIGS. 9 and 10, respectively.

(59) In addition, P. pemaquidensis survival was decreased from 96.6 to 91.8 percent after 72 hours of incubation and P. perurans survival was decreased from 92.1 to 90.2 percent 72 hours of incubation in mucus collected from fish fed Control 2″+A″ feed.

(60) It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb “comprise” and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article “a” or “an” preceding an element does not exclude the presence of a plurality of such elements.

(61) The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.