Method for treating or preventing bacterial disease in fish

Abstract

The present invention provides a method for treating or preventing a bacterial disease in fish, the method comprising intraperitoneally administering, to marine fish, an active ingredient selected from oxytetracycline or a salt thereof and doxycycline or a salt thereof.

Description

Method for treating or preventing bacterial diseases in fish

[0001] The present invention relates to a method for treating or preventing bacterial diseases in marine fish, particularly farmed fish.

[0002] Oxytetracycline (OTC), a tetracycline antibiotic, is commonly used in aquaculture both in Japan and overseas. In Japan, it is approved as an effective oral treatment for vibriosis and alpha-hemolytic streptococcosis in Perciformes, vibriosis in Tetraodontiformes, vibriosis, furunculosis, and alpha-hemolytic streptococcosis in Clupeiniformes, paracoccosis in Anguilliformes, and streptococcosis in Pleuronectiformes. Because OTC exhibits broad-spectrum antibacterial activity, it has been reported to be active against pathogens other than those mentioned above. Doxycycline is approved as an effective oral treatment for alpha-hemolytic streptococcosis in Perciformes.

[0003] Since around 2013, a mutant strain of alpha-hemolytic streptococcus (Lactococcus formosensis, hereafter also referred to as "type II streptococcus") that is resistant to conventional vaccines against streptococcus (Lactococcus garvieae) has been identified in fish such as yellowtail, causing widespread damage and becoming a problem. Vaccines against type II streptococcus have been developed, but problems have been pointed out, such as the insufficient duration of immunity compared to conventional vaccines. Furthermore, the use of antimicrobial agents in the fisheries sector requires appropriate use as veterinary drugs, including in the fisheries sector, in order to prevent the development of antimicrobial resistance, particularly to antibiotics used in humans.

[0004] It has been reported that the bioavailability of OTC when administered orally is low (Non-Patent Document 3), and to improve the efficacy of OTC, oral administration of OTC in combination with N-acetyl-D-glucosamine (Non-Patent Document 1) and oral administration of OTC together with an absorption improver (Patent Document 1) have been reported. Tetracycline-class antibacterial agents such as OTC have been reported to generate singlet oxygen, which causes oxidative stress in the body (Non-Patent Documents 4-6).

[0005] Nocardiosis, caused by the bacterium Nocardia seriolae, is one of the most serious bacterial diseases in aquaculture. Currently, no vaccines against nocardiosis are commercially available, and oral administration of sulfonamides is the common treatment. Nocardiosis, caused by Nocardia seriolae, is a major problem in yellowtail aquaculture. Edwardsiellosis, caused by Edwardsiella tarda, is a major problem in red sea bream and flounder aquaculture. Although OTC is effective against these pathogens in vitro, it has been reported that oral administration of OTC did not provide any therapeutic effect for these diseases (Non-Patent Documents 1 and 2).

[0006] Edwardsiellosis, a bacterial disease caused by Edwardsiella bacteria, is a problematic bacterial disease in red sea bream, flounder, and eels. The causative bacterium was previously thought to be Edwardsiella tarda, but analysis of fish-derived strains has led to the reclassification of red sea bream-derived strains as E. anguillarum and flounder-derived strains as E. piscicida. Edwardsiellosis can cause mass mortality in red sea bream aquaculture, with characteristic symptoms including head ulcers, abdominal distension, and skin abrasions. Edwardsiellosis, also known as paracolo disease in eels, is primarily caused by E. anguillarum, although both E. anguillarum and E. piscicida have been reported to be isolated from eel-derived strains. Another known Edwardsiella species is E. ictaluri, which is problematic in freshwater catfish and other fish. In Japan, E. ictaluri infection was confirmed in sweetfish (Ayu) in 2007, and the problem has expanded since then.

[0007] Epitheliocystis is a bacterial infection. The causative bacterium is thought to be a bacterium related to chlamydia, but no reports have been released on its identification. It is known to occur in red sea bream, as well as tiger pufferfish, carp, and amberjack. In red sea bream aquaculture, outbreaks in juvenile fish become a problem from the time of seedling introduction through the summer. Chlamydia bacteria (spherical or ovoid, 0.5-0.7 μm) infect gill epithelial cells, forming cysts (10-400 μm). While the presence of a small number of cysts in the gills is generally not a problem, severe cases can lead to mass mortality.

[0008] Patent Application No. 2019-172619

[0009] Akiyama, K., Morita, A., Hirazawa, N., Kawahara, H., 2018. The combination effect of N-acetyl-D-glucosamine on oral oxytetracycline treatment against Nocardia seriolae infection in the yellowtail Seriola quinqueradiata. Aquaculture 483, 149-153. Kanai, K., 2002. Therapeutic effect of sulfamonomethoxine and ormetoprim combination against Edwardsiellosis in Japanese flounder. Nagasaki University Faculty of Fisheries Research Reports 83, 1-4. Elema, MO, Hoff, KA, Kristensen, HG, 1996. Bioavailability of oxytetracycline from medicated feed administered to Atlantic salmon (Salmo salar L.) in seawater. Aquaculture 143, 7-14. Rok, J., Wrzesniok, D., Beberok, A. Otreba, M., Delijewski,M., & Buszman, E. (2018). Phototoxic effect of oxytetracycline on normal human melanocytes. Toxicology in Vitro, 48, 26-32; Yonar, ME, Yonar, SM, & Silici, S. (2011). Protective effect of propolis against oxidative stress and immunosuppression induced by oxytetracycline in rainbow trout (Oncorhynchus mykiss, W.). Fish & shellfish immunology, 31(2), 318-325; Hasan, T., & Khan, AU (1986).Phototoxicity of the tetracyclines: photosensitized emission of singlet delta dioxygen. Proceedings of the National Academy of Sciences, 83(13), 4604-4606.

[0010] According to one aspect of the present invention, there is provided a method for treating or preventing bacterial fish diseases such as type II alpha-hemolytic streptococcosis (hereinafter also referred to as "type II streptococcosis") or nocardiosis. According to another aspect of the present invention, there is provided a method for immunizing fish for preventing bacterial fish diseases such as type II streptococcosis or nocardiosis.

[0011] The present inventors have completed the present invention as a result of conducting experimental research on bacterial fish diseases, particularly type II streptococcosis or nocardiosis.

[0012] [A-1] A method for treating or preventing a bacterial fish disease, which comprises intraperitoneally administering to marine fish an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof.

[0013] [A-2] The method according to [A-1], wherein the marine fish is a fish of the order Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeidae, Salmoniformes, or Anguilliformes. [A-3] The method according to [A-1] or [A-2], wherein the marine fish is a fish of the order Perciformes, Seriformes.

[0014] [A-4] The method according to any one of [A-1] to [A-3], wherein the fish of the genus Seriola is selected from yellowtail (Seriola quinqueradiata), amberjack (Seriola dumerili), yellowtail amberjack (Seriola lalandi), long-finned amberjack (Seriola rivoliana), Seriola carpenteri, Seriola fasciata, southern amberjack (Seriola hippos), Seriola peruana, Seriola dorsalis, and Seriola zonata.

[0015] [A-5] The method according to any one of [A-1] to [A-4], wherein the marine fish is a yellowtail or an amberjack. [A-6] The method according to any one of [A-1] to [A-5], wherein the body weight of the marine fish to be intraperitoneally administered is 100 g or more.

[0016] [A-7] The method according to any one of [A-1] to [A-5], wherein the body weight of the marine fish to be intraperitoneally administered is 200 g or more. [A-8] The method according to any one of [A-1] to [A-5], wherein the body weight of the marine fish to be intraperitoneally administered is 250 g or more.

[0017] [A-9] The method according to any one of [A-1] to [A-8], wherein the body weight of the marine fish to be intraperitoneally administered is 1500 g or less. [A-10] The method according to any one of [A-1] to [A-8], wherein the body weight of the marine fish to be intraperitoneally administered is 1200 g or less.

[0018] [A-11] The method according to any one of [A-1] to [A-8], wherein the body weight of the marine fish to be intraperitoneally administered is 1000 g or less. [A-12] The method according to any one of [A-1] to [A-8], wherein the body weight of the marine fish to be intraperitoneally administered is 900 g or less.

[0019] [A-13] The method according to any one of [A-1] to [A-12], wherein the dosage of the active ingredient is 5 mg / kg or more of fish body weight. [A-14] The method according to any one of [A-1] to [A-13], wherein the dosage of the active ingredient is 10 mg / kg or more of fish body weight.

[0020] [A-15] The method according to any one of [A-1] to [A-14], wherein the dosage of the active ingredient is 15 mg / kg or more of fish body weight. [A-16] The method according to any one of [A-1] to [A-15], wherein the dosage of the active ingredient is 100 mg / kg or less of fish body weight.

[0021] [A-17] The method according to any one of [A-1] to [A-16], wherein the dosage of the active ingredient is 90 mg / kg or less of fish body weight. [A-18] The method according to any one of [A-1] to [A-17], wherein the dosage of the active ingredient is 80 mg / kg or less of fish body weight. [A-19] The method according to any one of [A-1] to [A-12], wherein the dosage of the active ingredient is 20 to 100 mg / kg of fish body weight.

[0022] [A-20] The method according to any one of [A-1] to [A-12], wherein the dosage of the active ingredient is 20 to 90 mg / kg of fish body weight. [A-21] The method according to any one of [A-1] to [A-12], wherein the dosage of the active ingredient is 20 to 80 mg / kg of fish body weight.

[0023] [A-22] The method according to any one of [A-1] to [A-12], wherein the dosage of the active ingredient is 20 to 70 mg / kg of fish body weight. [A-23] The method according to any one of [A-1] to [A-12], wherein the dosage of the active ingredient is 20 to 60 mg / kg of fish body weight.

[0024] [A-24] The method according to any one of [A-1] to [A-12], wherein the administered amount of the active ingredient is 20 to 50 mg / kg of fish body weight. [A-25] The method according to any one of [A-1] to [A-12], wherein the body weight of the marine fish to be intraperitoneally administered is 100 to 900 g and the administered amount of the active ingredient is 20 to 30 mg / kg of fish body weight.

[0025] [A-26] The method according to any one of [A-1] to [A-12], wherein the body weight of the marine fish to be intraperitoneally administered is 100 to 600 g, and the dose of the active ingredient is 20 to 60 mg / kg of body weight of the fish.

[0026] [A-27] The method according to any one of [A-1] to [A-26], wherein the bacterial fish disease is alpha-hemolytic streptococcosis or nocardiosis. [A-28] The method according to any one of [A-1] to [A-27], wherein the bacterial fish disease is streptococcosis or nocardiosis caused by a causative bacterium selected from Lactococcus garvieae, Lactococcus formosensis, and Nocardia seriolae.

[0027] [A-29] The method according to [A-27], wherein the α-hemolytic streptococcosis is type II α-hemolytic streptococcosis. [A-30] The method according to any one of [A-1] to [A-29], wherein the intraperitoneal administration is to a marine fish reared in a water temperature of 28°C or lower.

[0028] [A-31] The method according to [A-30], wherein the intraperitoneal administration is carried out to farmed fish kept in the sea at a time when seawater temperature is expected to rise. [A-32] The method according to any one of [A-1] to [A-31], wherein the intraperitoneal administration is carried out so that the blood concentration of oxytetracycline or doxycycline 24 hours after administration is 0.374 μg / ml or more.

[0029] [A-33] The method according to any one of [A-1] to [A-32], wherein oxytetracycline or doxycycline is administered intraperitoneally so that the blood concentration of oxytetracycline or doxycycline 48 hours after administration is 0.203 μg / ml or more.

[0030] [A-34] The method according to any one of [A-1] to [A-5], [A-13] to [A-24], and [A-27] to [A-33], wherein the body weight of the marine fish to be intraperitoneally administered is 100 g or more, 200 g or more, or 250 g or more, and 1500 g or less, 1200 g or less, 1000 g or less, or 900 g or less.

[0031] [A-35] The method according to any one of [A-1] to [A-12] and [A-27] to [A-34], wherein the dosage of the active ingredient is 5 mg / kg or more of fish body weight, 10 mg / kg or more of fish body weight, or 15 mg / kg or more of fish body weight, and 100 mg / kg or less of fish body weight, 90 mg / kg or less of fish body weight, or 80 mg / kg or less of fish body weight.

[0032] [A-36] The method according to any one of [A-1] and [A-6] to [A-34], wherein the marine fish is a fish belonging to the Sparidae family of the Sparidae order. [A-37] The method according to [A-36], wherein the fish belonging to the Sparidae family is selected from southern black porgy (Acanthopagrus sivicolus), Taiwan sea bream (Argyrops bleekeri Oshima), yellow sea bream (Evynnis tumifrons), crimson sea bream (Evynnis japonica), red sea bream (Pagrus major), black porgy (Acanthopagrus schlegelii), gilthead sea bream (Rhabdosargus sarba), and gilthead sea bream (Sparus aurata).

[0033] [A-38] The method according to any one of [A-1] to [A-26] and [A-29] to [A-37], wherein the bacterial fish disease is edwardsiellosis or epitheliocystis. [A-39] The method according to any one of [A-36] or [A-37], wherein the bacterial fish disease is edwardsiellosis caused by E. anguillarum. [A-40] The method according to any one of [A-36] or [A-37], wherein the bacterial fish disease is epitheliocystis caused by a chlamydiae bacterium.

[0034] [B-1] A composition comprising an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof, for use in the method according to any one of [A-1] to [A-40].

[0035] [C-1] A method for raising marine fish for food, which comprises treating or preventing bacterial fish diseases in marine fish by the method according to any one of [A-1] to [A-40].

[0036] [D-1] A method for immunizing marine fish against bacteria that cause bacterial fish diseases, comprising intraperitoneally administering to the marine fish an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof.

[0037] [D-2] The method according to [D-1], wherein the marine fish is a fish of the order Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeidae, Salmoniformes, or Anguilliformes. [D-3] The method according to [D-1] or [D-2], wherein the marine fish is a fish of the order Perciformes, Seriformes.

[0038] [D-4] The method according to any one of [D-1] to [D-3], wherein the fish of the genus Seriola is selected from yellowtail (Seriola quinqueradiata), amberjack (Seriola dumerili), yellowtail amberjack (Seriola lalandi), long-finned amberjack (Seriola rivoliana), Seriola carpenteri, Seriola fasciata, southern amberjack (Seriola hippos), Seriola peruana, Seriola dorsalis, and Seriola zonata.

[0039] [D-5] The method according to any one of [D-1] to [D-4], wherein the marine fish is a yellowtail or an amberjack. [D-6] The method according to any one of [D-1] to [D-5], wherein the body weight of the marine fish to be intraperitoneally administered is 100 g or more.

[0040] [D-7] The method according to any one of [D-1] to [D-5], wherein the body weight of the marine fish to be intraperitoneally administered is 200 g or more. [D-8] The method according to any one of [D-1] to [D-5], wherein the body weight of the marine fish to be intraperitoneally administered is 250 g or more.

[0041] [D-9] The method according to any one of [D-1] to [D-8], wherein the body weight of the marine fish to be intraperitoneally administered is 1500 g or less. [D-10] The method according to any one of [D-1] to [D-8], wherein the body weight of the marine fish to be intraperitoneally administered is 1200 g or less.

[0042] [D-11] The method according to any one of [D-1] to [D-8], wherein the body weight of the marine fish to be intraperitoneally administered is 1000 g or less. [D-12] The method according to any one of [D-1] to [D-8], wherein the body weight of the marine fish to be intraperitoneally administered is 900 g or less.

[0043] [D-13] The method according to any one of [D-1] to [D-12], wherein the dosage of the active ingredient is 5 mg / kg or more of fish body weight. [D-14] The method according to any one of [D-1] to [D-13], wherein the dosage of the active ingredient is 10 mg / kg or more of fish body weight.

[0044] [D-15] The method according to any one of [D-1] to [D-14], wherein the dosage of the active ingredient is 15 mg / kg or more of fish body weight. [D-16] The method according to any one of [D-1] to [D-15], wherein the dosage of the active ingredient is 100 mg / kg or less of fish body weight.

[0045] [D-17] The method according to any one of [D-1] to [D-16], wherein the dosage of the active ingredient is 90 mg / kg or less of fish body weight. [D-18] The method according to any one of [D-1] to [D-17], wherein the dosage of the active ingredient is 80 mg / kg or less of fish body weight. [D-19] The method according to any one of [D-1] to [D-12], wherein the dosage of the active ingredient is 20 to 100 mg / kg of fish body weight.

[0046] [D-20] The method according to any one of [D-1] to [D-12], wherein the dosage of the active ingredient is 20 to 90 mg / kg of fish body weight. [D-21] The method according to any one of [D-1] to [D-12], wherein the dosage of the active ingredient is 20 to 80 mg / kg of fish body weight.

[0047] [D-22] The method according to any one of [D-1] to [D-12], wherein the dosage of the active ingredient is 20 to 70 mg / kg of fish body weight. [D-23] The method according to any one of [D-1] to [D-12], wherein the dosage of the active ingredient is 20 to 60 mg / kg of fish body weight.

[0048] [D-24] The method according to any one of [D-1] to [D-12], wherein the administered amount of the active ingredient is 20 to 50 mg / kg of fish body weight. [D-25] The method according to any one of [D-1] to [D-12], wherein the body weight of the marine fish to be intraperitoneally administered is 100 to 900 g and the administered amount of the active ingredient is 20 to 30 mg / kg of fish body weight.

[0049] [D-26] The method according to any one of [D-1] to [D-12], wherein the body weight of the marine fish to be intraperitoneally administered is 100 to 600 g, and the dose of the active ingredient is 20 to 60 mg / kg of body weight of the fish.

[0050] [D-27] The method according to any one of [D-1] to [D-26], wherein the bacterial fish disease is alpha-hemolytic streptococcosis or nocardiosis. [D-28] The method according to any one of [D-1] to [D-27], wherein the bacterial fish disease is streptococcosis or nocardiosis caused by a causative bacterium selected from Lactococcus garvieae, Lactococcus formosensis, and Nocardia seriolae.

[0051] [D-29] The method according to [D-27], wherein the α-hemolytic streptococcosis is type II α-hemolytic streptococcosis. [D-30] The method according to any one of [D-1] to [D-29], wherein the intraperitoneal administration is to marine fish reared in a water temperature of 28°C or lower.

[0052] [D-31] The method according to [D-30], wherein the intraperitoneal administration is carried out to farmed fish kept in the sea at a time when seawater temperature is expected to rise. [D-32] The method according to any one of [D-1] to [D-31], wherein the intraperitoneal administration is carried out so that the blood concentration of oxytetracycline or doxycycline 24 hours after administration is 0.374 μg / ml or more.

[0053] [D-33] The method according to any one of [D-1] to [D-32], wherein oxytetracycline or doxycycline is administered intraperitoneally so that the blood concentration of oxytetracycline or doxycycline is 0.203 μg / ml or more 48 hours after administration. [D-34] The method according to any one of [D-1] to [D-33], wherein the marine fish are raised in an environment where they can be exposed to a causative bacterium of a bacterial fish disease before the intraperitoneal administration.

[0054] [D-35] The method according to any one of [D-1] to [D-34], wherein the marine fish are raised in a marine aquaculture pen where they can be exposed to bacteria that cause bacterial fish diseases present in the sea before intraperitoneal administration. [D-36] The method according to any one of [D-1] to [D-5], [D-13] to [D-24], and [D-27] to [D-35], wherein the body weight of the marine fish to be intraperitoneally administered is 100 g or more, 200 g or more, or 250 g or more, and 1500 g or less, 1200 g or less, 1000 g or less, or 900 g or less.

[0055] [D-37] The method according to any one of [D-1] to [D-12] and [D-27] to [D-36], wherein the dosage of the active ingredient is 5 mg / kg or more of fish body weight, 10 mg / kg or more of fish body weight, or 15 mg / kg or more of fish body weight, and 100 mg / kg or less of fish body weight, 90 mg / kg or less of fish body weight, or 80 mg / kg or less of fish body weight.

[0056] [D-38] The method according to any one of [D-1] and [D-6] to [D-37], wherein the marine fish is a fish belonging to the Sparidae family of the Sparidae order. [D-39] The method according to [D-38], wherein the fish belonging to the Sparidae family is selected from the group consisting of southern black porgy (Acanthopagrus sivicolus), Taiwan sea bream (Argyrops bleekeri Oshima), yellow sea bream (Evynnis tumifrons), crimson sea bream (Evynnis japonica), red sea bream (Pagrus major), black porgy (Acanthopagrus schlegelii), gilthead sea bream (Rhabdosargus sarba), and gilthead sea bream (Sparus aurata).

[0057] [D-40] The method according to any one of [D-1] to [D-26] and [D-29] to [D-39], wherein the bacterial fish disease is edwardsiellosis or epitheliocystis. [D-41] The method according to any one of [D-38] or [D-39], wherein the bacterial fish disease is edwardsiellosis caused by E. anguillarum. [D-42] The method according to any one of [D-38] or [D-39], wherein the bacterial fish disease is epitheliocystis caused by a chlamydiae bacterium.

[0058] [E-1] A composition comprising an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof, for use in the method according to any one of [D-1] to [D-42].

[0059] [F-1] A method for raising marine fish for food, which comprises treating or preventing bacterial fish diseases in marine fish by the method described in any one of [D-1] to [D-42].

[0060] [G-1] A method for treating or preventing a bacterial fish disease, which comprises intraperitoneally administering to marine fish an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof.

[0061] [G-2] The method according to [G-1], wherein the marine fish is a fish belonging to the order Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeidae, Salmoniformes, Anguilliformes, or Sparidae. [G-3] The method according to [G-1] or [G-2], wherein the marine fish is a fish of the order Perciformes, Seriidae, or family Sparidae.

[0062] [G-4] The method according to any one of [G-1] to [G-3], wherein the fish of the Seriidae family is selected from yellowtail (Seriola quinqueradiata), amberjack (Seriola dumerili), amberjack (Seriola lalandi), long-finned amberjack (Seriola rivoliana), Seriola carpenteri, Seriola fasciata, southern amberjack (Seriola hippos), Seriola peruana, Seriola dorsalis, and Seriola zonata, and the fish belonging to the Sparidae family is selected from southern black porgy (Acanthopagrus sivicolus), Taiwan sea bream (Argyrops bleekeri Oshima), yellow sea bream (Evynnis tumifrons), crimson sea bream (Evynnis japonica), red sea bream (Pagrus major), black porgy (Acanthopagrus schlegelii), gilthead sea bream (Rhabdosargus sarba), and gilthead sea bream (Sparus aurata).

[0063] [G-5] The method according to any one of [G-1] to [G-4], wherein the marine fish is red sea bream, black porgy, sea bream, gilthead seabream, yellowtail, or amberjack. [G-6] The method according to any one of [G-1] to [G-5], wherein the body weight of the marine fish to be intraperitoneally administered is 100 g or more, 200 g or more, or 250 g or more, and 1500 g or less, 1200 g or less, 1000 g or less, or 900 g or less.

[0064] [G-7] The method according to any one of [G-1] to [G-6], wherein the dosage of the active ingredient is 5 mg / kg or more of fish body weight, 10 mg / kg or more of fish body weight, or 15 mg / kg or more of fish body weight, and 100 mg / kg or less of fish body weight, 90 mg / kg or less of fish body weight, or 80 mg / kg or less of fish body weight.

[0065] [G-8] The method according to any one of [G-1] to [G-7], wherein the bacterial fish disease is alpha-hemolytic streptococcosis, nocardiosis, edwardsiellosis, or epitheliocystis.

[0066] [G-9] The method according to any one of [G-1] to [G-8], wherein the bacterial fish disease is streptococcosis or nocardiosis caused by a causative bacterium selected from Lactococcus garvieae, Lactococcus formosensis, and Nocardia seriolae; edwardsiellosis caused by E. anguillarum; or epitheliocystis caused by a chlamydiae bacterium.

[0067] [G-10] The method according to [G-8], wherein the α-hemolytic streptococcosis is type II α-hemolytic streptococcosis. [G-11] A composition comprising an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof, for use in the method according to any one of [G-1] to [G-10].

[0068] A method for raising marine fish for food, comprising treating or preventing bacterial fish diseases in marine fish by the method according to any one of [G-1] to [G-10].

[0069] [G-12] A method for immunizing marine fish against bacteria that cause bacterial fish diseases, comprising intraperitoneally administering to the marine fish an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof.

[0070] [H-1] A method for treating or preventing a bacterial fish disease, which comprises intraperitoneally or intramuscularly administering to marine fish an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof.

[0071] [H-2] The method according to [H-1], wherein the marine fish is a fish belonging to the order Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeidae, Salmoniformes, Anguilliformes, or Sparidae. [H-3] The method according to [H-1] or [H-2], wherein the marine fish is a fish of the order Perciformes, Seriidae, or family Sparidae.

[0072] [H-4] The method according to any one of [H-1] to [H-3], wherein the fish of the Seriidae family is selected from yellowtail (Seriola quinqueradiata), amberjack (Seriola dumerili), amberjack (Seriola lalandi), long-finned amberjack (Seriola rivoliana), Seriola carpenteri, Seriola fasciata, southern amberjack (Seriola hippos), Seriola peruana, Seriola dorsalis, and Seriola zonata, and the fish belonging to the Sparidae family is selected from southern black porgy (Acanthopagrus sivicolus), Taiwan sea bream (Argyrops bleekeri Oshima), yellow sea bream (Evynnis tumifrons), crimson sea bream (Evynnis japonica), red sea bream (Pagrus major), black porgy (Acanthopagrus schlegelii), gilthead sea bream (Rhabdosargus sarba), and gilthead sea bream (Sparus aurata).

[0073] [H-5] The method according to any one of [H-1] to [H-4], wherein the marine fish is a fish of the Sparidae family of the order Sparidae, and the active ingredient is administered intraperitoneally or intramuscularly. [H-6] The method according to any one of [H-1] to [H-5], wherein the body weight of the marine fish to be administered intraperitoneally or intramuscularly is 100 g or more, 200 g or more, or 250 g or more, and 1500 g or less, 1200 g or less, 1000 g or less, or 900 g or less.

[0074] [H-7] The method according to any one of [H-1] to [H-6], wherein the dosage of the active ingredient is 5 mg / kg or more of fish body weight, 10 mg / kg or more of fish body weight, or 15 mg / kg or more of fish body weight, and 100 mg / kg or less of fish body weight, 90 mg / kg or less of fish body weight, or 80 mg / kg or less of fish body weight.

[0075] [H-8] The method according to any one of [H-1] to [H-7], wherein the dosage of the active ingredient is 5 mg / kg or more, 10 mg / kg or less of fish body weight, and the bacterial fish disease is streptococcosis or nocardiosis caused by a causative bacterium selected from Lactococcus garvieae, Lactococcus formosensis, and Nocardia seriolae; edwardsiellosis caused by E. anguillarum; or epitheliocystis caused by a chlamydiae bacterium. [H-9] The method according to any one of [H-1] to [H-8], wherein the bacterial fish disease is streptococcosis or nocardiosis caused by a causative bacterium selected from Lactococcus garvieae, Lactococcus formosensis, and Nocardia seriolae; edwardsiellosis caused by E. anguillarum; or epitheliocystis caused by a chlamydiae bacterium.

[0076] [H-10] The method according to [H-8], wherein the α-hemolytic streptococcosis is type II α-hemolytic streptococcosis. [H-11] A composition comprising an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof, for use in the method according to any one of [H-1] to [H-10].

[0077] [H-12] A method for raising marine fish for food, comprising treating or preventing bacterial fish diseases in marine fish by the method according to any one of [H-1] to [H-10].

[0078] [I-1] A method for immunizing marine fish against bacteria that cause bacterial fish diseases, comprising intraperitoneally or intramuscularly administering to the marine fish an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof. [I-2] The method according to [I-1], wherein the marine fish is a fish belonging to the order Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeidae, Salmoniformes, Anguilliformes, or Sparidae. [I-3] The method according to [I-1] or [I-2], wherein the marine fish is a fish of the family Seriformes of the order Perciformes or the family Sparidae of the order Sparidae.

[0079] [I-4] The method according to any one of [I-1] to [I-3], wherein the fish of the Seriidae family is selected from yellowtail (Seriola quinqueradiata), amberjack (Seriola dumerili), amberjack (Seriola lalandi), long-finned amberjack (Seriola rivoliana), Seriola carpenteri, Seriola fasciata, southern amberjack (Seriola hippos), Seriola peruana, Seriola dorsalis, and Seriola zonata, and the fish of the Sparidae family is selected from southern black porgy (Acanthopagrus sivicolus), Taiwan sea bream (Argyrops bleekeri Oshima), yellow sea bream (Evynnis tumifrons), crimson sea bream (Evynnis japonica), red sea bream (Pagrus major), black porgy (Acanthopagrus schlegelii), gilthead sea bream (Rhabdosargus sarba), and gilthead sea bream (Sparus aurata).

[0080] [I-5] The method according to any one of [I-1] to [I-4], wherein the marine fish is a fish of the Sparidae family of the order Sparidae, and the active ingredient is administered intraperitoneally or intramuscularly. [I-6] The method according to any one of [I-1] to [I-5], wherein the body weight of the marine fish to be administered intraperitoneally or intramuscularly is 100 g or more, 200 g or more, or 250 g or more, and 1500 g or less, 1200 g or less, 1000 g or less, or 900 g or less.

[0081] [I-7] The method according to any one of [I-1] to [I-6], wherein the dosage of the active ingredient is 5 mg / kg or more of fish body weight, 10 mg / kg or more of fish body weight, or 15 mg / kg or more of fish body weight, and 100 mg / kg or less of fish body weight, 90 mg / kg or less of fish body weight, or 80 mg / kg or less of fish body weight.

[0082] [I-8] The method according to any one of [I-1] to [I-7], wherein the dosage of the active ingredient is 5 mg / kg or more, 10 mg / kg or less of fish body weight, and the bacterial fish disease is streptococcosis or nocardiosis caused by a causative bacterium selected from Lactococcus garvieae, Lactococcus formosensis, and Nocardia seriolae; edwardsiellosis caused by E. anguillarum; or epitheliocystis caused by a chlamydiae bacterium. [I-9] The method according to any one of [I-1] to [I-8], wherein the bacterial fish disease is streptococcosis or nocardiosis caused by a causative bacterium selected from Lactococcus garvieae, Lactococcus formosensis, and Nocardia seriolae; edwardsiellosis caused by E. anguillarum; or epitheliocystis caused by a chlamydiae bacterium.

[0083] [I-10] The method according to [I-8], wherein the α-hemolytic streptococcosis is type II α-hemolytic streptococcosis. [I-11] A composition comprising an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof, for use in the method according to any one of [I-1] to [I-10].

[0084] [I-12] A method for raising marine fish for food, comprising immunizing marine fish against bacteria that cause bacterial fish diseases by the method according to any one of [I-1] to [I-10].

[0085] In one aspect of the present invention, there is provided a method for treating and preventing bacterial fish diseases in marine fish that is highly safe for marine fish.In one aspect of the present invention, there is provided a method for immunizing marine fish against bacterial fish diseases that has a long-lasting effect.In one aspect of the present invention, there is provided a method for treating and preventing type II alpha-hemolytic streptococcosis or nocardiosis in marine fish, which are particularly problematic in farmed fish.In one aspect of the present invention, there is provided a method for treating and preventing edwardsiellosis or epitheliocystis in marine fish, which are particularly problematic in farmed fish.

[0086] In one aspect of the present invention, compared to conventional methods in which oxytetracycline or doxycycline is orally administered as an active ingredient, a method in which oxytetracycline or doxycycline is administered intraperitoneally or intramuscularly can reduce the amount of the active ingredient used and the risk of developing resistant bacteria.

[0087] Figure 1 is a graph showing the effect of intraperitoneal administration of OTC on type II streptococcosis (Example 2). Figure 2 is a graph showing the effect of intraperitoneal administration of OTC on nocardiosis (Example 3). Figure 3 is a graph showing the resistance of yellowtail to type II streptococcosis after treatment with intraperitoneal administration of OTC (Example 4). Figure 4 is a graph showing the resistance of yellowtail to nocardiosis after treatment with intraperitoneal administration of OTC (Example 5). Figure 5 is a graph showing the effect of intraperitoneal administration of OTC on Edwardsiellosis in red sea bream.

[0088] One aspect of the present invention provides a method for treating or preventing a bacterial fish disease, which comprises intraperitoneally administering to marine fish an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof.

[0089] One aspect of the present invention provides a method for treating or preventing bacterial fish diseases, which comprises intramuscularly administering to marine fish an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof.

[0090] In one aspect of the present invention, marine fish refers to fish that live in the sea. In one embodiment of the present invention, the marine fish are fish that develop bacterial infections, and are farmed fish or ornamental fish raised in fish pens or aquariums. In one aspect, the present invention is implemented in mass-reared farmed fish to prevent mass mortality due to bacterial infections. The present invention targets fish that are known to develop bacterial infections against which oxytetracycline or doxycycline is effective. Specifically, the target is fish belonging to the order Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeidae, Salmoniformes, or Anguilliformes. The target is fish belonging to the order Perciformes, Carangidae; the order Perciformes, Sparidae; the order Perciformes, Scombridae; the order Pleuronectiformes, Pleuronectidae; the order Tetraodontiformes, Tetraodontiformes; the order Anguilliformes, Anguilliformes. Specific examples include yellowtail, amberjack, amberjack and other yellowtail species, tuna, striped jack, flounder, red sea bream, pufferfish, salmon, trout, and eel.

[0091] Oxytetracycline (OTC) is a broad-spectrum tetracycline antibiotic with the structure (4S,4aR,5S,5aR,6S,12aS)-4-(dimethylamino-d)-3,5,6,10,11,12a-hexahydroxy-6-methyl-1,12-dioxo-1,4,4a,5,5a,6,12,12a-octahydrotetracene-2-carboxamide, used to treat bacterial infections. Oxytetracycline has a broad antibacterial spectrum and is effective against gram-positive and gram-negative bacteria, spirochetes, rickettsiae, and chlamydiae. It acts by inhibiting the synthesis of essential bacterial proteins. It is commercially available in bulk powder form, including OTC, OTC dihydrate (both lipid-soluble), and OTC hydrochloride (water-soluble). It is also sold as oxytetracycline alkyltrimethylammonium calcium salt. Both functions function as oxytetracycline in the body. The drugs approved for use in fisheries in Japan are hydrochloric acid (OTC), alkyltrimethylammonium calcium (OTC), and doxycycline.

[0092] Doxycycline is a broad-spectrum tetracycline antibiotic with the structure (4S,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide. Like OTC drugs, it is approved as a fishery drug in Japan. The following explanation will focus on oxytetracycline, but the same applies to doxycycline.

[0093] Unless otherwise specified, the dosage of the active ingredient herein is expressed as the amount of oxytetracycline or doxycycline salt administered converted into the amount of oxytetracycline or doxycycline. In one embodiment of the present invention, salts of oxytetracycline and doxycycline include acid addition salts such as hydrochlorides, sulfates, acetates, and nitrates.

[0094] The intraperitoneal administration described herein can be carried out by a method commonly used for fish, for example, using an injection needle and syringe. In one aspect of the present invention, intraperitoneal administration is carried out after anesthetizing the target fish. The anesthetic agent is not particularly limited as long as it is one commonly used for fish, and examples thereof include eugenol, 2-phenoxyethanol, and ethyl 3-aminobenzoate methanesulfonate. In one aspect of the present invention, intraperitoneal administration is carried out by injection.

[0095] The intramuscular administration described herein can be carried out by a method commonly used for fish, for example, using an injection needle and syringe. In one aspect of the present invention, intramuscular administration is carried out after anesthetizing the target fish. The anesthetic agent is not particularly limited as long as it is one commonly used for fish, and examples thereof include eugenol, 2-phenoxyethanol, and ethyl 3-aminobenzoate methanesulfonate. In one aspect of the present invention, intramuscular administration is carried out by injection.

[0096] In one aspect of the present invention, bacterial fish diseases include type I or type II alpha-hemolytic streptococcosis caused by Lactococcus garvieae or Lactococcus formosensis in yellowtail and tuna, nocardiosis caused by Nocardia seriolae in yellowtail and tuna, edwardsiellosis caused by Edwardsiella tarda in red seabream and flounder, and piscirickettsiosis caused by Piscirickettsia salmonis in salmonid fish. In one aspect of the present invention, bacterial fish diseases include edwardsiellosis caused by E. anguillarum or E. piscicida in sparid fish or flatfish, and epitheliocystis caused by Chlamydia species.

[0097] One aspect of the present invention is effective against diseases caused by intracellular parasitic bacteria. Examples of intracellular parasitic bacteria include Nocardia seriolae, the causative bacterium of nocardiosis, Edwardsiella tarda, the causative bacterium of edwardiella disease, and Mycobacterium bacteria, the causative bacterium of mycobacteriosis. Examples of intracellular parasitic bacteria include E. anguillarum or E. piscicida, the causative bacterium of edwardiella disease, and Chlamydia species, the causative bacterium of epitheliocystis disease. It is known that cellular immunity, rather than humoral immunity such as antibodies, plays an important role in fish defense against intracellular parasitic bacteria. Edwardsiella disease occurs in red sea bream and flounder. A decline in quality due to darkening of the body color caused by ultraviolet light has become a problem for farmed red sea bream. Therefore, fish ponds are shaded to maintain the color of farmed red sea bream closer to that of wild fish. Flounder farming is carried out in land-based facilities. Both fish species are cultivated in conditions that are less susceptible to sunlight, which is thought to reduce abrasion caused by photosensitivity, a side effect of OTC administration.

[0098] In one aspect of the present invention, a composition used in a method for treating, preventing, or immunizing against bacterial fish diseases comprises an active ingredient and an additive, such as a carrier, stabilizer, solvent, excipient, or diluent. In one embodiment of the present invention, the composition is a liquid formulation.

[0099] In one aspect of the present invention, the body weight of marine fish to be administered intraperitoneally is 40g or more, 50g or more, 100g or more, 200g or more, or 250g or more, and 1500g or less, 1200g or less, 1000g or less, 900g or less, or 600g or less, and is 40g or more to 1500g, 40g or more to 1200g, 40g or more to 1000g, 40g or more to 900g, 40g or more to 600g, 50g or more to 1500g, 50g or more to 1200g, 50g or more to 1000g, 50g or more to 50g 900g or less, 50g or more and 600g or less, 100g or more and 1500g or less, 100g or more and 1200g or less, 100g or more and 1000g or less, 100g or more and 900g or less, 100g or more and 600g or less, 200g or more and 1500g or less, 200g or more and 1200g or less, 200g or more and 1000g or less, 200g or more and 900g or less, 200g or more and 600g or less, 250g or more and 1500g or less, 250g or more and 1200g or less, 250g or more and 1000g or less, 250g or more and 900g or less, 250g or more and 600g or less.

[0100] In one aspect of the present invention, the body weight of marine fish to be administered intramuscularly is 40g or more, 50g or more, 100g or more, 200g or more, or 250g or more, and 1500g or less, 1200g or less, 1000g or less, 900g or less, or 600g or less, and is 40g or more to 1500g, 40g or more to 1200g, 40g or more to 1000g, 40g or more to 900g, 40g or more to 600g, 50g or more to 1500g, 50g or more to 1200g, 50g or more to 1000g, 50g or more to 50g 900g or less, 50g to 600g, 100g to 1500g, 100g to 1200g, 100g to 1000g, 100g to 900g, 100g to 600g, 200g to 1500g, 200g to 1200g, 200g to 1000g, 200g to 900g, 200g to 600g, 250g to 1500g, 250g to 1200g, 250g to 1000g, 250g to 900g, 250g to 600g. In one embodiment of the present invention, the marine fish is a yellowtail, particularly a yellowtail or an amberjack. In the case of intramuscular administration, any part of the fish's body can be administered safely, but the dorsal muscle can also be administered because it is thicker than other parts of the body and the risk of injury from injection is reduced.

[0101] In one aspect of the present invention, the dosage of the active ingredient in intraperitoneal administration is 5 mg / kg or more of fish body weight, 10 mg / kg or more of fish body weight, 15 mg / kg or more of fish body weight, 20 mg / kg or more of fish body weight, or 25 mg / kg or more of fish body weight, and 100 mg / kg or less of fish body weight, 90 mg / kg or less of fish body weight, 80 mg / kg or less of fish body weight, 70 mg / kg or less of fish body weight, 60 mg / kg or less of fish body weight, or 50 mg / kg or less of fish body weight, and 5 mg / kg or more of fish body weight to 100 mg / kg or 5 mg / kg or more of fish body weight to 90 mg / kg or less of fish body weight. Lower, 5 mg / kg fish weight or more and 80 mg / kg fish weight or less, 5 mg / kg fish weight or more and 70 mg / kg fish weight or less, 5 mg / kg fish weight or more and 60 mg / kg fish weight or less, 5 mg / kg fish weight or more and 50 mg / kg fish weight or less, 10 mg / kg fish weight or more and 100 mg / kg g fish weight or less, 10 mg / kg fish weight or more and 90 mg / kg fish weight or less, 10 mg / kg fish weight or more and 80 mg / kg fish weight or less, 10 mg / kg fish weight or more and 70 mg / kg fish weight or less, 10 mg / kg fish weight or more and 60 mg / kg fish weight or less, 10 mg / kg fish weight or less Above 50 mg / kg fish weight or less, 15 mg / kg fish weight or more and 100 mg / kg fish weight or less, 15 mg / kg fish weight or more and 90 mg / kg fish weight or less, 15 mg / kg fish weight or more and 80 mg / kg fish weight or less, 15 mg / kg fish weight or more and 70 mg / kg fish weight or less, 15 mg / kg fish weight or more, 60 mg / kg fish weight or less, 15 mg / kg fish weight or more, 50 mg / kg fish weight or less, 20 mg / kg fish weight or more, 100 mg / kg fish weight or less, 20 mg / kg fish weight or more, 90 mg / kg fish weight or less, 20 mg / kg fish weight or more, 80 mg / kg less than 20 mg / kg of fish weight but not exceeding 70 mg / kg of fish weight, more than 20 mg / kg of fish weight but not exceeding 60 mg / kg of fish weight, more than 20 mg / kg of fish weight but not exceeding 50 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 100 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 90 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 80 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 70 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 60 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 50 mg / kg of fish weight.

[0102] The dosage here refers to the amount of oxytetracycline and doxycycline, and when salts thereof are used, the amount is expressed in terms of oxytetracycline or doxycycline.

[0103] In one aspect of the present invention, the dosage of the active ingredient in intramuscular administration is 5 mg / kg or more of fish body weight, 10 mg / kg or more of fish body weight, 15 mg / kg or more of fish body weight, 20 mg / kg or more of fish body weight, or 25 mg / kg or more of fish body weight, and 100 mg / kg or less of fish body weight, 90 mg / kg or less of fish body weight, 80 mg / kg or less of fish body weight, 70 mg / kg or less of fish body weight, 60 mg / kg or less of fish body weight, or 50 mg / kg or less of fish body weight, and 5 mg / kg or more of fish body weight to 100 mg / kg or 5 mg / kg or more of fish body weight to 90 mg / kg or less of fish body weight. Lower, 5 mg / kg fish weight or more and 80 mg / kg fish weight or less, 5 mg / kg fish weight or more and 70 mg / kg fish weight or less, 5 mg / kg fish weight or more and 60 mg / kg fish weight or less, 5 mg / kg fish weight or more and 50 mg / kg fish weight or less, 10 mg / kg fish weight or more and 100 mg / kg g fish weight or less, 10 mg / kg fish weight or more and 90 mg / kg fish weight or less, 10 mg / kg fish weight or more and 80 mg / kg fish weight or less, 10 mg / kg fish weight or more and 70 mg / kg fish weight or less, 10 mg / kg fish weight or more and 60 mg / kg fish weight or less, 10 mg / kg fish weight or less Above 50 mg / kg fish weight or less, 15 mg / kg fish weight or more and 100 mg / kg fish weight or less, 15 mg / kg fish weight or more and 90 mg / kg fish weight or less, 15 mg / kg fish weight or more and 80 mg / kg fish weight or less, 15 mg / kg fish weight or more and 70 mg / kg fish weight or less, 15 mg / kg fish weight or more, 60 mg / kg fish weight or less, 15 mg / kg fish weight or more, 50 mg / kg fish weight or less, 20 mg / kg fish weight or more, 100 mg / kg fish weight or less, 20 mg / kg fish weight or more, 90 mg / kg fish weight or less, 20 mg / kg fish weight or more, 80 mg / kg less than 20 mg / kg of fish weight but not exceeding 70 mg / kg of fish weight, more than 20 mg / kg of fish weight but not exceeding 60 mg / kg of fish weight, more than 20 mg / kg of fish weight but not exceeding 50 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 100 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 90 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 80 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 70 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 60 mg / kg of fish weight, more than 25 mg / kg of fish weight but not exceeding 50 mg / kg of fish weight.The dosage here refers to the amount of oxytetracycline and doxycycline, and when salts thereof are used, the amount is expressed in terms of oxytetracycline or doxycycline.

[0104] In one aspect of the present invention, the marine fish are farmed fish, and the water temperature is 16°C or higher, 17°C or higher, 18°C ​​or higher, 19°C or higher, 20°C or higher, 21°C or higher, or 22°C or higher, and 28°C or lower, 27°C or lower, 26°C or lower, or 25°C or lower, 16°C or higher and 28°C or lower, 16°C or higher and 27°C or lower, 16°C or higher and 26°C or lower, 16°C or higher and 25°C or lower, 17°C or higher and 28°C or lower, 17°C or higher and 27°C or lower, 17°C or higher and 26°C or lower, 17°C or higher and 25°C or lower, 18°C ​​or higher and 28°C or lower , 18°C ​​or higher and 26°C or lower, 18°C ​​or higher and 25°C or lower, 19°C or higher and 28°C or lower, 19°C or higher and 27°C or lower, 19°C or higher and 26°C or lower, 19°C or higher and 25°C or lower, 20°C or higher and 28°C or lower, 20°C or higher and 27°C or lower, 20°C or higher and 26°C or lower, 20°C or higher and 25°C or lower, 21°C or higher and 28°C or lower, 21°C or higher and 27°C or lower, 21°C or higher and 26°C or lower, 21°C or higher and 25°C or lower, 22°C or higher and 28°C or lower, 22°C or higher and 27°C or lower, 22°C or higher and 26°C or lower, or 22°C or higher and 25°C or lower. In one embodiment of the present invention, intraperitoneal administration is performed in autumn, winter, or from spring to summer when seawater temperatures are rising.

[0105] In one aspect of the present invention, the marine fish are farmed fish, and the water temperature is 16°C or higher, 17°C or higher, 18°C ​​or higher, 19°C or higher, 20°C or higher, 21°C or higher, or 22°C or higher, and 28°C or lower, 27°C or lower, 26°C or lower, or 25°C or lower, 16°C or higher and 28°C or lower, 16°C or higher and 27°C or lower, 16°C or higher and 26°C or lower, 16°C or higher and 25°C or lower, 17°C or higher and 28°C or lower, 17°C or higher and 27°C or lower, 17°C or higher and 26°C or lower, 17°C or higher and 25°C or lower, 18°C ​​or higher and 28°C or lower , 18°C ​​or higher and 26°C or lower, 18°C ​​or higher and 25°C or lower, 19°C or higher and 28°C or lower, 19°C or higher and 27°C or lower, 19°C or higher and 26°C or lower, 19°C or higher and 25°C or lower, 20°C or higher and 28°C or lower, 20°C or higher and 27°C or lower, 20°C or higher and 26°C or lower, 20°C or higher and 25°C or lower, 21°C or higher and 28°C or lower, 21°C or higher and 27°C or lower, 21°C or higher and 26°C or lower, 21°C or higher and 25°C or lower, 22°C or higher and 28°C or lower, 22°C or higher and 27°C or lower, 22°C or higher and 26°C or lower, 22°C or higher and 25°C or lower. In one embodiment of the present invention, intramuscular administration is performed in autumn, winter, or from spring to summer when seawater temperatures are rising.

[0106] In one aspect of the present invention, the marine fish are fish cultivated in aquariums on land. In another aspect of the present invention, the marine fish are fish cultivated in fish cages on the sea surface.

[0107] In one aspect, the method of the present invention may further include rearing the marine fish under a light-shielded condition for a certain period of time after intraperitoneal administration. One aspect of the present invention provides a method for treating or preventing bacterial fish diseases, comprising intraperitoneally administering to marine fish an active ingredient selected from oxytetracycline or a salt thereof and doxycycline or a salt thereof, and rearing the marine fish under a light-shielded condition for a certain period of time after administration. In one embodiment of the present invention, rearing the marine fish under a light-shielded condition for a certain period of time after intraperitoneal administration can suppress side effects caused by photosensitivity reactions. Examples of rearing under a light-shielded condition include rearing in an indoor aquarium or a marine fish pen protected from sunlight. The rearing period under a light-shielded condition can be determined by a person skilled in the art of the present invention, and examples include 6 hours, 12 hours, 24 hours, 2 days, 3 days, and 1 week. In one embodiment of the present invention, the rearing period under light-blocking conditions is, for example, until the blood drug concentration becomes 0.8 μg / ml or less, 0.6 μg / ml or less, 0.5 μg / ml or less, 0.3 μg / ml or less, 0.2 μg / ml or less, or 0.1 μg / ml or less.

[0108] In one aspect, the method of the present invention may further include rearing the marine fish under a light-shielded condition for a certain period of time after intramuscular administration. One aspect of the present invention provides a method for treating or preventing bacterial fish diseases, comprising intramuscularly administering an active ingredient selected from oxytetracycline or a salt thereof and doxycycline or a salt thereof to marine fish, and rearing the marine fish under a light-shielded condition for a certain period of time after administration. In one embodiment of the present invention, rearing the marine fish under a light-shielded condition for a certain period of time after intramuscular administration can suppress side effects caused by photosensitivity reactions. Examples of rearing under a light-shielded condition include rearing in an indoor aquarium or a marine fish pen protected from sunlight. The rearing period under a light-shielded condition can be determined by a person skilled in the art of the present invention, and examples include 6 hours, 12 hours, 24 hours, 2 days, 3 days, and 1 week. In one embodiment of the present invention, the rearing period under light-blocking conditions is, for example, until the blood drug concentration becomes 0.8 μg / ml or less, 0.6 μg / ml or less, 0.5 μg / ml or less, 0.3 μg / ml or less, 0.2 μg / ml or less, or 0.1 μg / ml or less.

[0109] In one aspect, the method of the present invention may further comprise administering an antioxidant to the marine fish in combination with the intraperitoneal administration. In one embodiment of the present invention, an antioxidant is administered to the marine fish before, after, or simultaneously with the intraperitoneal administration. In one embodiment of the present invention, the antioxidant is administered intraperitoneally, intramuscularly, or orally. Examples of antioxidants include vitamin A, vitamin C, tocopherol, ethoxyquin, dibutylhydroxytoluene, and butylhydroxyanisole. The dosage of the antioxidant can be determined appropriately by one skilled in the art.

[0110] In one aspect, the method of the present invention may further comprise administering an antioxidant to marine fish in combination with intramuscular administration. In one embodiment of the present invention, an antioxidant is administered to marine fish before, after, or simultaneously with the intramuscular administration. In one embodiment of the present invention, the antioxidant is administered intraperitoneally, intramuscularly, or orally. Examples of antioxidants include vitamin A, vitamin C, tocopherol, ethoxyquin, dibutylhydroxytoluene, and butylhydroxyanisole. The dosage of the antioxidant can be determined appropriately by one skilled in the art.

[0111] According to one method of the present invention, there is provided a method for immunizing marine fish against bacteria that cause bacterial fish diseases, comprising intraperitoneally administering to the marine fish an active ingredient selected from oxytetracycline or a salt thereof and doxycycline or a salt thereof. In one embodiment of the present invention, prior to the intraperitoneal administration, the marine fish are raised in a natural environment, such as in a marine fish pen, in an environment where they can be exposed to the bacteria that cause bacterial fish diseases. In another embodiment of the present invention, the intraperitoneal administration is carried out after it has been confirmed that a bacterial fish disease has occurred in an area close to the marine fish pen where the target marine fish are kept.

[0112] According to one method of the present invention, there is provided a method for immunizing marine fish against bacteria that cause bacterial fish diseases, comprising intramuscularly administering to the marine fish an active ingredient selected from oxytetracycline or a salt thereof and doxycycline or a salt thereof. In one embodiment of the present invention, prior to the intramuscular administration, the marine fish are raised in a natural environment, such as in a marine fish pen, in an environment where they can be exposed to the bacteria that cause bacterial fish diseases. In another embodiment of the present invention, the intramuscular administration is carried out after it has been confirmed that a bacterial fish disease has occurred in an area close to the marine fish pen where the target marine fish are kept.

[0113] In one aspect of the present invention, side effects resulting from administration of the active ingredient are confirmed by a symptom called "chafing" in marine fish. In one embodiment, "chafing" is an abnormality such as discoloration, inflammation, or damage in the dorsal skin tissue of marine fish, and can be confirmed, for example, by visually inspecting the dorsal area of ​​marine fish in a fish preserve or aquarium. Examples of the present invention are described below, but the present invention is not limited thereto.

[0114] Example 1: Effects of OTC Administration on Yellowtail - Materials and Methods: A study was conducted to assess the effects of rearing environment, fish weight at the time of administration, and administration dose on the side effects of intraperitoneal OTC administration. For the study, yellowtail fry produced from fertilized eggs in an indoor land-based rearing facility were used. The fry were raised to an appropriate weight for the study using a commercially available EP feed (product name: Otohime, Nisshin Marubeni Feed Co., Ltd.) and then housed in a 500-liter indoor tank or a 2-ton outdoor tank. Four trials were conducted, and detailed conditions for each trial, including rearing conditions, number of test fish, fish size, OTC dose, and water temperature, are shown in Table 1. After each OTC administration, yellowtail were housed in a single tank for each test. The water exchange rate for the tank was 83.3% per hour. Test fish housed in outdoor tanks were allowed an acclimation period of at least one week between their placement and the start of the study to allow them to fully acclimate to the environment. After habituation, the fish were anesthetized with FA100 (Bussan Animal Health Co., Ltd.) and administered OTC hydrochloride (OTC Injection 10% "Fujita", Fujita Pharmaceutical Co., Ltd.) intraperitoneally. The administered dose (shown as the amount of OTC) is shown in Table 1, and the amount of OTC administered was determined by measuring the weight of each fish at the time of administration. After administration, the fish were observed for 14 or 15 days, and their condition and whether or not they died were observed and recorded. Test fish that died during the test period were removed from the tank and observed.

[0115]

[0116] Results and Discussion: In indoor Test 1, no abnormalities or deaths were observed in yellowtail at any dose. However, deaths were observed in outdoor Tests 2–4 (Table 2). The average fish weight was 291.3 g in Test 2 and 560.3 g in Test 3. In both tests, mortality was observed in the OTC 100 mg / kg fish weight group. In Test 4, the average fish weight was 900.4 g, and 60% of the fish died in the OTC 50 mg / kg fish weight group. All dead fish exhibited obvious dorsal abrasions. Mortality was not observed indoors, but only in outdoor tests. Therefore, intraperitoneal administration of OTC clearly caused adverse effects outdoors. Furthermore, dorsal abrasions were commonly observed in dead fish, suggesting that sunlight may be involved in the adverse effects. Furthermore, comparing the results of Tests 2–4, larger fish exhibited higher mortality, indicating that they were more susceptible to adverse effects from OTC administration. These results suggest that intraperitoneal administration of OTC to yellowtail in outdoor environments can cause side effects depending on the dose, and that the larger the fish, the more likely the side effects are to occur. For yellowtail weighing less than 900 g, intraperitoneal administration of OTC at a dose of 25 mg / kg body weight or less is unlikely to cause side effects.

[0117]

[0118] Example 2: Examination of OTC Dosage for Treatment of Type II Streptococcus Infection Materials and Methods Example 1 demonstrated that side effects could be avoided in yellowtail weighing less than 900g if OTC was administered at a dose of 25 mg / kg of fish body weight or less. Therefore, we investigated whether administering OTC at a dose of 25 mg / kg of fish body weight or less is effective in treating type II streptococcosis, a common disease in yellowtail. We used yellowtail fry produced from fertilized eggs in an indoor land-based breeding facility. 107 fry were housed in two 500L tanks. The test fish were anesthetized with 2-phenoxyethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and the causative bacterium of type II streptococcosis (Lactococcus formosensis, isolated from diseased yellowtail) was infused at 1.0 x 10 per fish. 5 The test fish were infected by intraperitoneal injection of cells. The onset of disease in the test group was confirmed by the appearance of dead or abnormally swimming fish. 21 hours after infection, 24 test fish, or 22.4% of the total, were found dead or had abnormal swimming behavior. Therefore, OTC was administered intraperitoneally. OTC hydrochloride (OTC Injection 10% "Fujita" (Fujita Pharmaceutical Co., Ltd.) was used as the OTC formulation, and the doses (listed as OTC) were 6.25, 12.5, 25, and 50 mg / kg of fish body weight. When administering the 6.25, 12.5, and 25 mg / kg of fish body weight, the undiluted formulation was diluted appropriately with saline because the volume of the administered solution was very small. The negative control group received an intraperitoneal injection of 0.1 ml of saline. The number of fish in each group was 16 for the 6.25 and 50 mg groups, 12.5, and 25 mg groups. There were 17 fish in each group, one for the 100 mg dose group and one for the control group. Test fish given OTC intraperitoneally were housed in 200L tanks for each dose and reared for 24 days. Test fish that died during the rearing period were necropsied and confirmed to be exhibiting one of the following typical symptoms of type II streptococcosis: gill anemia, pericarditis, or caudal peduncle ulcers. During the test period, the water exchange rate in the tanks was 83.3% per hour, and the feeding rate was 2% of the fish's body weight per day. The average water temperature in the rearing water was 24.3°C, with a range of 24.0-24.9°C.

[0119] Results and Discussion The test results are shown in Table 3 below and Figure 1.

[0120]

[0121] The cumulative mortality rate in the negative control group, where saline was administered intraperitoneally, was 100% (Figure 1). In contrast, the cumulative mortality rates in the intraperitoneally administered OTC groups were 0% for the 6.25 mg and 50 mg groups, 29.4% for the 12.5 mg group, and 23.5% for the 25 mg group, significantly lower than those in the negative control group. These results demonstrate that intraperitoneal administration of OTC at doses of 6.25 mg / kg body weight or higher is effective in treating type II streptococcosis in yellowtail. Therefore, OTC doses in the range of 6.25–25 mg / kg body weight were effective in treating this disease in yellowtail weighing less than 900 g without adverse effects.

[0122] Example 3: Examination of the intraperitoneal dose of OTC for the treatment of fish infected with nocardiosis Materials and Methods: The therapeutic effect of intraperitoneal administration of OTC on nocardiosis, a common disease of yellowtail, along with type II streptococcosis, was examined. For the test, fry produced from fertilized yellowtail eggs in an indoor land-based breeding facility were used. 100 fry with an average weight of 51.4 g were housed in four 200 L tanks, 25 in each. After acclimatization for 10 days, the fish were infected with Nocardia seriolae, the causative bacterium of nocardiosis. For infection, the water supply to each tank was stopped and 1.7 x 10 pre-cultured Nocardia seriolae was added. 3 The test was conducted by adding the OTC to a concentration of 100 CFU / ml and rearing the fish under static conditions for one hour. Water flow was resumed one hour after addition. Onset of symptoms in the test group was confirmed by the appearance of dead fish exhibiting symptoms characteristic of nocardiosis. 14 days after experimental infection, dead fish were observed in one tank, and characteristic symptoms of nocardiosis, such as nodules on the gills and body surface, were confirmed. The following day, 15 days after infection, OTC was administered intraperitoneally. OTC hydrochloride (OTC Injection 10% "Fujita," Fujita Pharmaceutical Co., Ltd.) was used as the injection formulation. Three OTC treatment groups were administered at doses of 12.5 mg / kg, 25 mg / kg, and 50 mg / kg of fish body weight. The negative control group was not administered OTC and the fish continued to be reared as is. After OTC administration, the fish were reared for 39 days, and the number of dead fish in each tank was recorded. Dead fish were removed from the tank and necropsied, confirming the presence of nodules on the gills and body surface, characteristic symptoms of nocardiosis. During the test period, the daily feed intake was 1.5% of the fish's body weight from the introduction of the 200L tank until experimental infection, 3.5% of the fish's body weight from experimental infection until 30 days after infection, and 3% of the fish's body weight from 32 days after infection onwards. The rearing water temperature ranged from 19.4 to 23.8°C, with an average of 22.3°C.

[0123] Results and Discussion The test results are shown in Table 4 below and Figure 2.

[0124]

[0125]

[0126] The cumulative mortality rate in the negative control group at the end of the study was 60.0% (Figure 2). Mortality in the OTC 12.5 mg / kg fish body weight group tended to be delayed compared to the negative control group, but by the end of the study, the cumulative mortality rate had reached 58.0%. Meanwhile, the cumulative mortality rates in the OTC 25 mg and OTC 50 mg / kg fish body weight groups were 28.0% and 12.0%, respectively, which were significantly lower than those in the negative control group. These results demonstrate that intraperitoneal administration of OTC at doses of 25 mg / kg fish body weight or higher is effective in treating nocardiosis in yellowtail.

[0127] Example 4: Examination of whether fish treated with OTC acquire immunity to type II streptococcosis Materials and Methods For the test, fry produced from fertilized yellowtail eggs in an indoor land-based breeding facility were used. Sixty fry with an average weight of 78.1 g were housed in three 200 L tanks, 20 fish per tank. Two of the tanks had a density of 5.0 × 10 per fish. 3 Experimental infection was performed by intraperitoneally administering a Lactococcus formosensis bacterial suspension. Because some fish died 24 hours after experimental infection, the surviving fish were given OTC intraperitoneally at 50 mg / kg body weight. After this, the fish were reared for 7 weeks after infection, and 15 surviving fish were collected from both tanks and combined into one 200L tank. The remaining tank was maintained without experimental infection, but was administered OTC at 50 mg / kg body weight at the same time as the two tanks mentioned above (negative control). The injection formulation used was OTC hydrochloride (OTC Injection 10% "Fujita", Fujita Pharmaceutical Co., Ltd.). The feeding rate was 1% of fish body weight during maintenance. Water temperature was maintained between 23.6 and 25.1°C. Seven weeks after OTC administration, 15 yellowtail (average fish weight 107.1 g) from each of the two groups mentioned above were given 5.0 × 10 of OTC per fish. 3 The fish were infected by intraperitoneally administering a Lactococcus formosensis suspension of cells. They were kept for 14 days after infection, and the number of dead fish in each tank was recorded. Dead fish were confirmed by necropsy to be showing one of the following symptoms characteristic of type II streptococcosis: gill anemia, pericarditis, or caudal peduncle ulcers. After experimental infection, the feeding rate was set at 2% of the fish's body weight per day. After infection, the water temperature was maintained at 22.3-24.5°C.

[0128] Results and Discussion The test results are shown in Table 5 below and Figure 3.

[0129]

[0130] The mortality rate after reinfection in the group treated with intraperitoneal OTC (50 mg / kg body weight) 24 hours after infection with type II streptococcosis was significantly lower than that in the negative control group, which received intraperitoneal OTC at the same time as treatment (Fig. 3, P < 0.05). The mortality rate in the control group treated with intraperitoneal OTC was 100%, suggesting that the high survival rate in the treated group was not due to residual OTC. Therefore, it was demonstrated that yellowtail treated with intraperitoneal OTC acquired resistance to the disease.

[0131] Example 5: Immunization of Yellowtail Treated for Nocardiosis by Intraperitoneal OTC Administration. Materials and Methods: Fish treated for nocardiosis by intraperitoneal OTC administration (Example 3) at 25 mg and 50 mg / kg body weight were used in this study. Thirty-nine days after OTC treatment, surviving test fish from each group were placed in 500-L tanks. The average fish weights at the time of placement were 165.9 g for the 25 mg treatment group and 169.2 g for the 50 mg treatment group. Additionally, as a negative control, yellowtail (average fish weight: 155.1 g) that had been intraperitoneally administered 50 mg / kg body weight of OTC on the same day as Group 2, where OTC was administered to treat nocardiosis, were placed in a separate 500-L tank. After two days of acclimation, each test fish was experimentally infected with Nocardia seriolae using the same method as in Example 3. The experimental infection concentration of Nocardia seriolae at this time was 3.5 × 10 3CFU / ml. Each test group was infected with 20 fish in the control group, 17 in the 25 mg treatment group, and 20 in the 50 mg treatment group. After experimental infection, the fish were reared for 30 days, and the number of dead fish in each tank was recorded. Test fish that died during the experimental period were necropsied and confirmed to have symptoms characteristic of nocardiosis. After experimental infection, the feeding rate was 3.5% of fish body weight only one day after infection, but because food intake decreased due to fish weight gain, it was reduced to 3% of fish body weight per day from two days onwards. The rearing water temperature in each tank was maintained within a range of 23.3 to 25.2°C, with an average of 24.4°C.

[0132] Results and Discussion The test results are shown in Table 6 below and Figure 4.

[0133]

[0134] The cumulative mortality rate in the negative control group was 95% (Figure 4). In contrast, the cumulative mortality rates in the OTC treatment groups were 29% for the 25 mg / kg fish body weight group and 5% for the 50 mg / kg fish body weight group. The 95% mortality rate in the negative control group treated with OTC suggests that the high survival rate in the treatment group was not due to residual OTC. Therefore, it was demonstrated that yellowtail treated for nocardiosis by intraperitoneal OTC administration acquired resistance to the disease.

[0135] As described above, it was revealed that intraperitoneal administration of OTC in outdoor environments can cause side effects that can result in deaths of fish depending on the dose and the size of the fish inoculated. By setting a dose that does not cause these side effects, it has become clear that it is possible to treat fish bacterial diseases caused by bacteria that are susceptible to OTC.

[0136] [Example 6] Efficacy of OTC injection against Edwardsiellosis in red sea bream Materials and methods Red sea bream juveniles derived from artificial seedlings that had been reared in a marine fish pen were transported to a land-based facility and then housed in five 200 L tanks, 15 fish per tank (average fish weight: 49 g). 8Experimental infection with E. anguillarum was performed by intraperitoneal injection of Edwardsiella anguillarum NRIA-631 strain (obtained through the Fisheries Genetic Resources Conservation Project of the National Research and Development Agency, Fisheries Science and Technology Agency) at CFU / fish equivalent. Four hours after experimental infection, four of the five groups received intraperitoneal injections of OTC injection (Fujita 10% OTC Injection, Fujita Pharmaceutical Co., Ltd.) at 6.25, 12.5, 25, and 50 mg / kg body weight, respectively. Fish were anesthetized with 2-phenoxyethanol in seawater at 250 ppm. The remaining group served as a non-medicated control. Starting the day after experimental infection, fish were fed a diet equivalent to 1% of their body weight daily, and mortality was monitored for up to 7 days after experimental infection. On the final day, all surviving fish were removed, anesthetized as described above, and necropsied. Bacteria were isolated from the kidneys using a platinum loop and SS agar medium (Shimadzu Diagnostics Co., Ltd.). If any fish died, bacteria were isolated from the kidneys on SS agar medium and Gram-stained smears were prepared, and if black colonies characteristic of E. anguillarum and Gram-negative bacilli were observed, the deaths were considered to be due to Edwardsiellosis. The rearing water temperature during the test period remained at 25.4 to 25.7°C.

[0137] Results and Discussion The results are shown in Table 7 and FIG.

[0138]

[0139] In the untreated control group, 13 of 15 fish died of Edwardsiellosis. In contrast, no fish died in the OTC injection group, except for one dead fish 7 days after infection in the 25 mg / kg fish body weight group (Figure 5). Mortality in the 25 mg / kg fish body weight group was due to cannibalism, and E. anguillarum was not isolated. Bacteria were not isolated from the kidneys of surviving fish 7 days after experimental infection in the 25 and 50 mg / kg fish body weight groups. OTC injection is also effective against Edwardsiellosis in red sea bream, at least within the range of 6.25 to 50 mg / kg fish body weight tested in this study. Furthermore, at doses above 25 mg / kg fish body weight, bacteria were not isolated from surviving fish, demonstrating its efficacy.

[0140] Example 7: Efficacy of OTC Injection against Epitheliocystis in Red Seabream. Materials and Methods: A batch containing red seabream derived from artificially hatched fish naturally infected with epitheliocystis in a marine fish pen was identified by microscopic observation of gills. Fish from the same batch (average fish weight 45 g) were transported to a land-based facility and housed in five 200 L tanks (15 fish per tank). Four of the five tanks were then intraperitoneally administered OTC injections (Fujita 10% OTC Injection, Fujita Pharmaceutical Co., Ltd.) at concentrations of 6.25, 12.5, 25, and 50 mg / kg fish weight, respectively. Fish were anesthetized with seawater containing 2-phenoxyethanol at 250 ppm. Starting the day after administration, the fish were fed daily with feed equivalent to 1% of their body weight. Eight days after OTC administration, four red sea bream from each group were taken and the number of epitheliocystis cysts formed on the gill filaments of the first gill arch of the left gill was counted under a microscope (100x magnification). The water temperature during the experiment was kept between 25.3 and 25.8°C.

[0141] Results and Discussion The results are shown in Table 8.

[0142]

[0143] The average cyst count in the untreated control group was 57.5. In contrast, in the OTC injection group, the average cyst count in the group treated with 6.25 mg / kg fish body weight was 0.25, and no cysts were observed in the groups treated with 12.5, 25, or 50 mg / kg BW OTC injections. These results demonstrate that OTC injection is effective against epitheliocystis, at least within the range of 6.25 to 50 mg / kg BW tested in this study.

Claims

1. A method for treating or preventing bacterial fish diseases, which comprises intraperitoneally administering to marine fish an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof.

2. The method according to claim 1, wherein the marine fish is a fish belonging to the order Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeidae, Salmoniformes, Anguilliformes or Sparidae.

3. The method according to claim 1 or 2, wherein the marine fish is a fish of the order Perciformes, genus Seriola, or the order Sparidae.

4. The method according to any one of claims 1 to 3, wherein the fish of the Seriidae family is selected from yellowtail (Seriola quinqueradiata), amberjack (Seriola dumerili), amberjack (Seriola lalandi), long-finned amberjack (Seriola rivoliana), Seriola carpenteri, Seriola fasciata, southern amberjack (Seriola hippos), Seriola peruana, Seriola dorsalis, and Seriola zonata, and the fish of the Sparidae family is selected from southern black porgy (Acanthopagrus sivicolus), orange sea bream (Argyrops bleekeri Oshima), yellow sea bream (Evynnis tumifrons), crimson sea bream (Evynnis japonica), red sea bream (Pagrus major), black porgy (Acanthopagrus schlegelii), gilthead sea bream (Rhabdosargus sarba), and gilthead sea bream (Sparus aurata).

5. The method according to any one of claims 1 to 4, wherein the marine fish is red sea bream, black porgy, sea bream, European gilthead sea bream, yellowtail or amberjack.

6. A method according to any one of claims 1 to 5, wherein the body weight of the marine fish to be administered intraperitoneally is 100g or more, 200g or more, or 250g or more, and 1500g or less, 1200g or less, 1000g or less, or 900g or less.

7. The method according to any one of claims 1 to 6, wherein the dosage of the active ingredient is 5 mg / kg or more of fish body weight, 10 mg / kg or more of fish body weight, or 15 mg / kg or more of fish body weight, and 100 mg / kg or less of fish body weight, 90 mg / kg or less of fish body weight, or 80 mg / kg or less of fish body weight.

8. The method according to any one of claims 1 to 7, wherein the bacterial fish disease is alpha-hemolytic streptococcosis, nocardiosis, edwardsiellosis or epitheliocystis.

9. The method according to any one of claims 1 to 8, wherein the bacterial fish disease is streptococcosis or nocardiosis caused by a causative bacterium selected from Lactococcus garvieae, Lactococcus formosensis, and Nocardia seriolae; edwardsiellosis caused by E. anguillarum; or epitheliocystis caused by a chlamydiae bacterium.

10. The method of claim 8, wherein the alpha-hemolytic streptococcosis is type II alpha-hemolytic streptococcosis.

11. A composition comprising active ingredients selected from oxytetracycline or a salt thereof and doxycycline or a salt thereof, for use in the method according to any one of claims 1 to 10.

12. A method for raising marine fish for food, comprising treating or preventing bacterial fish diseases in marine fish by the method according to any one of claims 1 to 10.

13. A method for immunizing marine fish against bacteria that cause bacterial fish diseases, which comprises intraperitoneally administering to the marine fish an active ingredient selected from oxytetracycline or a salt thereof, and doxycycline or a salt thereof.

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