Methods for controlling intracellular parasitic bacterial infections in farmed fish

Abstract

To provide a method for controlling intracellular parasitic bacterial infections in cultured fish, in which repeated administration of an immunostimulant in combination with an antibiotic maximizes the efficacy of the antibiotic and minimizes the onset of intracellular parasitic bacterial infections with a short dosing period.SOLUTION: An antibiotic is administered at predetermined intervals together with feeding of an immunostimulant to young-of-the-year cultured fish. By repeatedly performing both natural infection with target intracellular parasitic bacteria and treatment after infection multiple times, the young-of-the-year cultured fish acquire immunity against the intracellular parasitic bacteria, thereby suppressing the onset of intracellular parasitic bacterial infections in two-year-old cultured fish.SELECTED DRAWING: None

Description

Technical Field

[0003] , , , , ,

[0001] The present invention relates to a method for controlling intracellular parasitic bacterial infections in farmed fish, which can minimize the mortality of farmed fish until shipment and the disposal at the time of shipment due to intracellular parasitic bacterial infections.

Background Art

[0002] In terms of the landing volume of farmed fish by fish species in Japan, sea bream (red sea bream) ranks second after yellowtail, and red sea bream farming is a very important industry. However, in recent years, the damage caused by fish diseases has been on the increase trend. Among them, the damage amount due to Edwardsiella disease (also called Tarda disease) of farmed red sea bream was 760 million yen in Reiwa元年 and 520 million yen in Reiwa 2 years (Ministry of Agriculture, Forestry and Fisheries: Information on the occurrence status of fish disease damage). Especially in areas with a high risk of Edwardsiella disease, there are also fishing grounds where the number of shipped fish is less than 50% of the number of introduced fry due to the mortality during growth and the disposal of infected fish at the time of shipment. Edwardsiella disease is an infectious disease of farmed fish caused by the infection of bacteria belonging to the genus Edwardsiella, which are Gram-negative bacilli (Edwardsiella tarda, E. piscicida, E. anguillarum), and occurs throughout the year regardless of the fish age. Since bacteria belonging to the genus Edwardsiella are intracellular parasitic bacteria, they tend to become chronic, leading to a decrease in the shipment volume of farmed fish such as red sea bream and flounder. So far, countermeasures against infectious diseases of farmed fish often rely on vaccines. In fact, inactivated vaccines have been put into practical use for many diseases and have achieved great effects. Therefore, in (Patent Document 1), a vaccine using inactivated cells of an Edwardsiella tarda-derived strain has been studied. In addition, regarding the prevention and treatment of Edwardsiella disease, preventive and therapeutic agents and preventive and therapeutic methods using crude drugs (Patent Document 2) and fish feed containing medium-chain fatty acids having antibacterial activity (Patent Document 3) have been studied.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

[0004] Here, immunity refers to the defense function against pathogens such as viruses or bacteria (a mechanism that protects the body from pathogens), and the vaccines currently used to combat infectious diseases in farmed fish apply the principle of this immune response. To explain in more detail, when farmed fish are infected with a certain pathogen such as a virus or bacteria, the immune cells in the body remember the information of the pathogen, and resistance to subsequent infections increases, thus protecting the body from that pathogen through the immune response. However, since Edwardsiella bacteria multiply within the cells of living organisms, they cannot be controlled by inactivated vaccines. In contrast, there are research reports suggesting that control is only possible with live vaccines, but since sea bream farming often takes place in seawater, using live vaccines is virtually impossible. As a control method other than vaccination, the use of antibiotics can be considered. Traditionally, fosfomycin (trade name Fosmicin) has been approved as an aquatic drug for the treatment of edwaziella disease in perciform fish, but as mentioned above, serious damage is still occurring, and it is reasonable to conclude that sufficient therapeutic effects have not been achieved. For the reasons stated above, no effective drugs or treatments for edvaziella disease have yet been found to be satisfactory. In particular, there is a strong desire from those involved in sea bream farming for the development of effective preventive (and therapeutic) methods for edvaziella disease, which causes significant economic losses. This is a challenge common not only to edvaziella disease but to intracellular parasitic bacterial infections in general. This invention has been made in view of the above circumstances, and aims to provide a method for controlling intracellular parasitic bacterial infections in farmed fish, which maximizes the effectiveness of antibiotics by using an immunostimulant and an antibiotic in combination and administering them repeatedly, thereby minimizing the onset of intracellular parasitic bacterial infections with short-term administration. [Means for solving the problem]

[0005] The present invention provides a method for controlling intracellular parasitic bacterial infections in farmed fish that is in line with the aforementioned objective. This method involves administering antibiotics to juvenile farmed fish at predetermined intervals in conjunction with the administration of an immunostimulant, thereby repeating natural infection with the target intracellular parasitic bacteria and subsequent treatment multiple times. This process allows the juvenile farmed fish to acquire immunity to the intracellular parasitic bacteria and suppresses the onset of intracellular parasitic bacterial infections in 2-year-old farmed fish. Here, we define fish in their first year of captivity as "yearling fish," and those from January of the following year onward as "two-yearling fish." To briefly explain the theory behind the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, it applies the principle of vaccines mentioned earlier by intentionally creating periods of no medication to allow farmed fish to be infected with intracellular parasitic bacteria, and then repeating these periods of medication treatment to increase the resistance of farmed fish to infection. However, it is empirically known that administering antibiotics alone after the onset of infection makes it difficult to eradicate all bacteria already proliferating in the body, and some bacteria are carried over into the farmed fish. Therefore, in order to minimize bacterial carryover, it is important to administer immunostimulants and antibiotics in combination. To date, there have been no reports of improvement in performance (farming yield) by using immunostimulants and antibiotics in combination in farmed fish. Immunostimulants and antibiotics are administered to all yearlings in the aquaculture system, and there is no need to select out any aquaculture fish that have developed intracellular parasitic bacterial infections (infection with intracellular parasitic bacteria) at the time of administration. It is preferable to administer immunostimulants continuously (daily) to young farmed fish during the period when they are susceptible to infection by the target intracellular parasitic bacteria (the period when the target intracellular parasitic bacteria are thought to be highly active). The interval between antibiotic administrations should be selected as appropriate.

[0006] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, the interval between administration of the antibiotic in combination with the immunostimulant is 1 week to 3 months (more preferably 4 weeks to 8 weeks), which is the period for allowing the farmed young fish to be naturally infected with the intracellular parasitic bacteria. Here, it is preferable to perform (repeat) multiple cycles in which a single medication period is basically 3 to 15 days (more preferably 5 to 10 days), and the interval between medications is 1 week to 3 months (more preferably 4 weeks to 8 weeks).

[0007] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, the antibiotic is preferably a tetracycline antibiotic.

[0008] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, the antibiotic is more preferably doxycycline.

[0009] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, the immunostimulant is preferably Ascophyllum nodosum extract. Ascophyllum nodosum is a type of large brown algae that grows in the coastal areas of Northern Europe and is used as livestock feed and a plant growth stimulant. Ascophyllum nodosum contains amino acids, vitamins, ash, polyphenols, and various minerals, as well as water-soluble polysaccharides such as alginic acid, fucoidan, mannitol, laminarin, and ascophyllan. Ascophyllan is a polysaccharide unique to Ascophyllum nodosum and is similar to fucoidan in that it contains L-fucose and sulfate groups, but its sugar residue composition is different, and it is known to have apoptosis-inducing ability and antitumor activity against certain cancer cells. Preventive and therapeutic compositions using Ascophyllum nodosum extract may contain Ascophyllum nodosum itself in any form, such as dried powder, but may also contain one or more compounds isolated from Ascophyllum nodosum. Any known method can be used to isolate the compounds contained in Ascophyllum nodosum, but solvent extraction is a preferred method. Ascophyllum nodosum used as the extraction raw material can be from any source. Since the raw algae have a high water content, it is preferable to use dried and powdered algae, which have been washed to remove salt, as the extraction raw material. The average composition of the dried Ascophyllum nodosum powder used as the extraction raw material is 5-10% protein, 45-60% carbohydrates, 17-20% ash, 2-4% lipids, and 10-12% moisture. As a pretreatment, the dried Ascophyllum nodosum powder may be treated with an acidic aqueous solution to remove acid-soluble components. The extraction of compounds from Ascophyllum nodosum is carried out, for example, using 10 to 100 times (by mass) the amount of Ascophyllum nodosum dry powder in water. When the water containing Ascophyllum nodosum dry powder is stirred at room temperature (around 15 to 25°C) for 12 to 24 hours, ascophyllan is extracted into the water along with alginic acid, etc. If necessary, more water may be added and the extraction may be carried out for another 12 to 24 hours at around 100°C. The crude extract obtained in this way contains other polysaccharides, proteins, carbohydrates, lipids, ash, minerals, pigments, polyphenols, etc. None of these are harmful to humans or animals, so when adding Ascophyllum nodosum extract to food or feed, it may be used as is without separation.

[0010] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, the immunostimulant preferably contains one or more of lactic acid bacteria, ascophyllan, and β-glucan.

[0011] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, it is preferable to administer antibiotics once when the intracellular parasitic bacteria surviving in the body of the two-year-old farmed fish begin to become active again.

[0012] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, the antibiotic administered to the two-year-old farmed fish is preferably doxycycline or oxytetracycline.

[0013] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, the farmed fish may be fish belonging to the orders Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeformes, Salmoniformes, or Anguilliformes.

[0014] The present invention provides a method for controlling intracellular parasitic bacterial infections in farmed fish, and is preferably applicable to edwaziella disease in red sea bream, edwaziella disease in flounder, nocardiosis in the genus Seriola of the order Perciformes, mycobacterium disease in the genus Seriola of the order Perciformes, and paracolo disease (edwaziella disease) in eels. [Effects of the Invention]

[0015] The present invention provides a method for controlling intracellular parasitic bacterial infections in farmed fish. By repeatedly administering an immunostimulant and an antibiotic in combination during the juvenile stage of farmed fish, the synergistic effect enhances immunity, minimizing mortality among juvenile fish and further minimizing the incidence of intracellular parasitic bacterial infections in 2-year-old fish before shipment. Since all fish possess an immune system, this method can be applied to all fish species.

[0016] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, when the interval between administration of antibiotics in combination with an immunostimulant is 1 week to 3 months (more preferably 4 weeks to 8 weeks), which is the period for natural infection of young farmed fish with intracellular parasitic bacteria, it is possible to effectively utilize natural infection to maximize the effectiveness of the antibiotics and reduce the amount of medication administered.

[0017] In the method for controlling intracellular parasitic bacterial infections in cultured fish according to the present invention, when the antibiotic is a tetracycline antibiotic, a high therapeutic effect can be obtained at a low cost.

[0018] In the method for controlling intracellular parasitic bacterial infections in cultured fish according to the present invention, when the antibiotic is doxycycline, it has high permeability into cells and has a high therapeutic effect against intracellular parasitic bacteria that grow inside cells.

[0019] In the method for controlling intracellular parasitic bacterial infections in cultured fish according to the present invention, when the immunostimulant is an extract of Ascophyllum nodosum, due to the synergistic effect with the antibiotic, the number of intracellular parasitic bacteria can be suppressed to a minimum. Thereby, it is possible to reduce the mortality rate in fry, prevent the recurrence of intracellular parasitic bacterial infections, and prevent the carry-over of intracellular parasitic bacteria from fry to two-year-old fish.

[0020] In the method for controlling intracellular parasitic bacterial infections in cultured fish according to the present invention, when the immunostimulant contains any one or more of lactic acid bacteria, ascophyllan, and β-glucan, due to the synergistic effect with the antibiotic, the number of intracellular parasitic bacteria can be suppressed to a minimum. Thereby, it is possible to reduce the mortality rate in fry, prevent the recurrence of intracellular parasitic bacterial infections, and prevent the carry-over of intracellular parasitic bacteria from fry to two-year-old fish.

[0021] In the method for controlling intracellular parasitic bacterial infections in cultured fish according to the present invention, when the antibiotic is administered once at the time when the intracellular parasitic bacteria surviving in the body of two-year-old cultured fish start to become active again, the onset of intracellular parasitic bacterial infections in two-year-old fish can be more effectively prevented.

[0022] In the method for controlling intracellular parasitic bacterial infections in cultured fish according to the present invention, when the antibiotic administered to two-year-old cultured fish is doxycycline or oxytetracycline, a low mortality rate is maintained until shipment.

[0023] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, if the farmed fish belong to the orders Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeformes, Salmoniformes, or Anguilliformes, various types of farmed fish can be protected from infections caused by intracellular parasitic bacteria.

[0024] The present invention provides a method for controlling intracellular parasitic bacterial infections in farmed fish. When applied to diseases such as edwaziella disease in sea bream, edwaziella disease in flounder, nocardiosis in species of the genus Seriola, mycobacterium disease in species of the genus Seriola, and paracolo disease in eels, it can reduce overall losses in the aquaculture industry, regardless of the species of farmed fish. [Brief explanation of the drawing]

[0025] [Figure 1] This diagram shows the protocol for a clinical trial to confirm the therapeutic effect of antibiotics used to treat edwaziella disease in farmed sea bream. [Figure 2] This figure shows the results of a clinical trial to confirm the therapeutic effect of antibiotics used to treat edwaziella disease in farmed sea bream. [Figure 3] This figure shows the results of a test to confirm the immune-acquiring effect of an immunostimulant used to treat edwaziella disease in farmed sea bream. [Figure 4] This figure shows the protocol for a confirmation study of the optimal drug administration method for treating edwaziella disease in farmed sea bream. [Figure 5] This figure shows the results of a study confirming the synergistic effect of administering Ascophyllum nodosum extract and antibiotics against nocardiosis in yellowtail. [Figure 6] This figure shows the results of a study confirming the synergistic effect of administering Ascophyllum nodosum extract and antibiotics against mycobacterial disease in yellowtail. [Figure 7] This figure shows the results of a study confirming the synergistic effect of administering lactic acid bacteria and antibiotics to yellowtail tuna to treat nocardiosis. [Modes for carrying out the invention]

[0026] Next, with reference to the attached drawings, embodiments of the present invention will be described to facilitate understanding of the present invention. A method for controlling intracellular parasitic bacterial infections in farmed fish according to one embodiment of the present invention involves repeatedly administering an immunostimulant and antibiotics in combination to young fish, thereby increasing acquired immunity to the target bacteria. This minimizes the remaining bacteria in the fish after administration, preventing the recurrence of intracellular parasitic bacterial infections, including Edwardsiella disease. Furthermore, it aims to reduce mortality and discarding of fish at the time of shipment without administering medication to two-year-old fish.

[0027] In previous fish disease control efforts, including those for Edwardsiella disease, the cause was identified and medication was started only after deaths were observed. However, this method of medication often resulted in delays in administration. While the number of deaths decreased temporarily after the medication was discontinued, recurrences occurred after the drug's effect wore off, requiring repeated medication. This suggests that even when the number of deaths drops to zero after medication, there is a high probability that intracellular parasitic bacteria remain in the fish's bodies. As a result, this leads to increased production costs due to the increased amount of medication used and an increase in the cumulative number of deaths, causing difficulties for fish farmers. In particular, while mortality in two-year-old fish is a problem in farmed sea bream with edwaziella disease, it is believed that the actual infection occurs in one-year-old fish. This is because even if one-year-old fish are infected with edwaziella bacteria, the activity of the edwaziella species temporarily stops during the winter, so the number of mortality in one-year-old fish does not increase significantly, and it appears as if the infection has subsided. However, in reality, the edwaziella species is carried over, and then, in early spring, the activity of the edwaziella species becomes active, causing edwaziella disease in two-year-old fish.

[0028] Therefore, in order to suppress the onset of Edwardsiella disease in two-year-old fish, countermeasures are necessary while the fish are still young. Specifically, it is considered necessary to enhance the immunity of young farmed sea bream against Edwardsiella by administering immunostimulants and antibiotics at prescribed intervals to young fish (farmed sea bream) during aquaculture, thereby repeating natural infection with Edwardsiella and subsequent treatment multiple times. Here, the interval between administrations of the immunostimulant and antibiotic is preferably 1 week to 3 months (more preferably 4 weeks to 8 weeks), but is not limited thereto. Furthermore, the single dose, number of doses, and total dose of each immunostimulant and antibiotic can be selected as appropriate.

[0029] As for antibiotics, tetracycline antibiotics are preferred, and doxycycline, in particular, is preferred due to its high intracellular permeability and effectiveness against intracellular parasitic bacteria, but it is not limited to this. For example, drugs that were previously considered ineffective in aquaculture farms despite the bacteria being susceptible to the drug may not have been effective simply because they were administered in the late stages of infection; they are likely to be effective if administered early in the infection. Furthermore, by administering drugs with an immunotherapy-centered approach, the range of available drugs expands significantly, and the problem of drug-resistant bacteria can be overcome. Specifically, oxolinic acid, sulfonamides in general, florfenicol, and fosfomycin are used. For example, in the treatment of edwaziella disease in farmed sea bream, doxycycline is typically administered for 3 to 15 days (more preferably 5 to 10 days) in a single dose, and it is preferable to perform this procedure multiple times. It is also desirable to administer it at a rate of 20 to 400 mg / kg·BW (more preferably 40 to 80 mg / kg·BW) per day. Furthermore, while Ascophyllum nodosum extract is preferred as an immunostimulant, it is not limited thereto. For example, an immunostimulant derived from beta-glucan or lactic acid bacteria may be used.

[0030] As described above, by repeatedly administering immunostimulants and antibiotics to farmed sea bream juveniles, it is believed that if immunity is acquired during the juvenile stage, the onset of Edwardsiella disease can be effectively suppressed even when the fish reach two years of age. However, from winter in the yearling fish until around May in the two-year-old fish, the activity of the remaining Edwardsiella bacteria in the body weakens, and at the same time, the immune function also declines. Therefore, if no preventative measures are taken when Edwardsiella bacteria become active in early spring, there is a risk that two-year-old fish will develop Edwardsiella disease before being shipped. To this end, administering antibiotics to two-year-old fish once when Edwardsiella bacteria become active (around April to July) can more reliably suppress the onset of Edwardsiella disease. The antibiotic administered at this time can be either doxycycline or oxytetracycline. From a cost perspective, oxytetracycline is preferable to doxycycline because it is cheaper, but it is not limited to these two.

[0031] In this embodiment, the focus has been on edwaziella disease in farmed sea bream, but since all fish possess immune mechanisms, the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention can be applied to all fish species. For example, it can be applied to edwaziella disease in flounder, nocardiosis in the genus Seriola in the order Perciformes, mycobacterium disease in the genus Seriola in the order Perciformes, and paracolo disease (edwaziella disease) in eels, all caused by the same intracellular parasitic bacteria. [Examples]

[0032] The following describes examples taken to confirm the effects of the present invention, but these examples do not limit the technical scope of the present invention in any way.

[0033] <Tests to confirm the therapeutic effect of antibiotics> A study was conducted to verify the therapeutic (preventive) effect of an antibiotic selected as a treatment for edwaziella disease in farmed sea bream. The study protocol is shown in Figure 1.

[0034] (Test Section 1) In a farming raft (8m x 8m x 5m) where approximately 5,000 two-year-old farmed sea bream (average weight 1kg) were being cultivated, doxycycline, an antibiotic, was administered orally (by feeding with a spreading agent) after deaths due to Edwardsiella disease were observed. The dosage was 80mg per kg of body weight per dose, administered for 5 consecutive days, with a 14-day rest period (dosing interval) in between, for a total of 3 doses.

[0035] (Examination Section 2) The treatment was the same as in Study Group 1, except that the single dose was 40 mg per kg of body weight and the number of doses was 10 consecutive days.

[0036] (Control groups 1 and 2) In control groups 1 and 2 (one aquaculture raft each), the conditions were the same as in experimental group 1, except that no medication was administered.

[0037] Figure 2 shows the cumulative number of deaths due to edwaziella disease in experimental groups 1 and 2 and control groups 1 and 2. A comparison between experimental groups 1 and 2 and control groups 1 and 2 revealed the therapeutic effect of doxycycline (an antibiotic) on edwaziella disease. Furthermore, a comparison between experimental group 1 and experimental group 2 revealed that even with the same total dose of doxycycline, the cumulative number of deaths in experimental group 2 was lower than in experimental group 1. This indicates that administering low concentrations of doxycycline over a long period, as in experimental group 2, yields a higher therapeutic effect than administering high concentrations of doxycycline over a short period, as in experimental group 1. However, even after repeated administration of doxycycline, the number of deaths never dropped to zero, and the cumulative number of deaths continued to increase. This confirmed that starting doxycycline administration in fish that are two years old is not cost-effective.

[0038] <Confirmation of therapeutic effects of immunostimulants> A study was conducted to confirm the immune-boosting effect of an immunostimulant against edwaziella disease in farmed sea bream.

[0039] (Examination Section 3) Approximately 10,000 farmed sea bream (average weight 150g) were administered to aquaculture rafts (8m x 8m x 5m) containing 10,000 young fish. The antibiotic doxycycline and the immunostimulant Ascophyllum nodosum extract were administered. Doxycycline was administered at a dose of 40mg per kg of body weight for 10 days, with a 45-day interval between doses to allow for natural infection, for a total of two doses. Ascophyllum nodosum extract was administered at a dose of 4mg per kg of body weight, daily for a total of 90 doses from September to November.

[0040] (Control group 3) In control group 3, the treatment was the same as in test group 3, except that Ascophyllum nodosum extract was not administered.

[0041] Figure 3 shows the changes in the number of deaths due to Edwardsiella disease in experimental group 3 and control group 3. A comparison between test group 3 and control group 3 revealed a synergistic effect against edwaziella disease when Ascophyllum nodosum extract (an immunostimulant) was used in combination with antibiotics. Bacteriostatic antibiotics are highly effective at inhibiting bacterial activity, but have low bactericidal effects when used alone. However, it is believed that their bactericidal effect is enhanced when used in combination with immunostimulants through synergistic action with the immune system. Therefore, it is thought that the combined use of Ascophyllum nodosum extract not only reduces the number of deaths but also, through a synergistic effect, reduces the number of Edwardsiella bacteria in the body, thus preventing the carryover of Edwardsiella bacteria from juvenile fish to 2-year-old fish.

[0042] For the farmed sea bream in experimental plot 3 and control plot 3, a portion was separated in February when they were two years old (experimental plot 3: 4,620 fish, control plot 3: 4,340 fish), and the cumulative number of deaths until shipment was then checked. As shown in the lower part of Figure 3, the final yield from rearing to shipment (number of fish at shipment / number of fish at rearing × 100) was 85.8% in control group 3, but 93.3% in experimental group 3. This suggests that while doxycycline alone administered to juvenile fish has a limited preventive effect on disease development in 2-year-old fish, its combination with an immunostimulant provides a sustained preventive effect even in 2-year-old fish, leading to improved performance (yield). Furthermore, while the yield is normally around 70%, it increased to approximately 85% to 93% when the juvenile fish acquired resistance to infection, demonstrating the importance of acquiring resistance to infection in the juvenile fish.

[0043] <Confirmation of Lifetime Medication Schedule> Based on the above test results, a trial was conducted to determine the optimal medication method from the introduction to the shipment of farmed sea bream. Approximately 10,000 sea bream were farmed in each farming raft (cage) (8m x 8m x 5m), and the average weight of the juvenile farmed sea bream at the start of farming was 30g. Medication was administered on a raft-by-raft basis, as in the above trial, and the trial period was approximately two years. The trial protocol is shown in Figure 4.

[0044] (Comparative Example 1: No medication administered) Three aquaculture rafts (fish pens) each raised 10,000 farmed sea bream without medication from the start of farming in June until February of their second year (during their first year). Then, 5,000 fish were randomly selected from each group and divided into two fish pens, where they were farmed without medication until October of the following year. The cumulative number of deaths due to Edwardsiella disease was then investigated from July to September, when mortality from Edwardsiella disease increases in the two-year-old fish.

[0045] (Comparative Example 2: Conventional Measures) In the same three aquaculture rafts (fish pens) as in Comparative Example 1, 5,000 farmed sea bream were randomly selected from those raised without medication from the start of farming in June until February of their second year of age, and divided into two fish pens. They were farmed without medication until deaths due to Edwardsiella disease were confirmed around June of their second year of age. In July, doxycycline was administered in 10-day courses, with a single dose of 40 mg per kg of body weight, for a total of three courses. At this time, no reduction in the number of deaths was observed, so the drug-free period (drug interval) was set to 5 days. Note that this drug-free period was not intended to build immunity, but is the same as the current general drug administration method of administering medication only after deaths are observed.

[0046] (Example 1) To confirm the synergistic effect of medication, 4 mg of Ascophyllum nodosum extract per kg of body weight was administered daily (approximately 150 times) from June 15, the start of cultivation, to 10,000 juvenile farmed sea bream raised in each of five aquaculture rafts (cages). In July, doxycycline was administered in 10-day courses of 40 mg per kg of body weight, for a total of three courses. During this time, a drug-free period of approximately 45 days was provided to allow natural infection with Edwardsiella bacteria. 5,000 farmed sea bream were randomly selected from the 2-year-old fish and divided into two aquaculture rafts (cages), where they were then farmed without medication.

[0047] (Example 2) In Example 2, 5,000 farmed sea bream were randomly selected from the same five farming rafts (cages) used in Example 1, where they were raised in the same manner as the first year of life until they became two-year-old fish. These 5,000 fish were then distributed among the five farming rafts (cages). In June (the time when Edwardsiella bacteria begin to become active), the fish were administered the antibiotic oxytetracycline once. Thus, Example 2 differs from Example 1 in that the two-year-old fish were administered the antibiotic (oxytetracycline) once in June.

[0048] Table 1 shows the cumulative number of deaths due to Edwardsiella disease in 2-year-old fish in Comparative Examples 1 and 2 and Examples 1 and 2.

[0049] [Table 1]

[0050] Table 1 shows that in Comparative Example 1, where no measures were taken to prevent Edwardsiella disease from the start of cultivation to shipment (cultivation was carried out without medication), more than 500 fish died during the period from July to September, which is the peak period for the onset of Edwardsiella disease in 2-year-old fish. In Comparative Example 2, the number of deaths was reduced to about half that of Comparative Example 1. However, it was found that, similar to conventional fish disease control measures, administering doxycycline (an antibiotic) after the onset of symptoms was not sufficiently effective for larger 2-year-old fish.

[0051] In Example 1, it was found that by administering Ascophyllum nodosum extract daily to young fish to enhance their immunity, while simultaneously administering doxycycline (an antibiotic), the incidence of edwaziella disease in 2-year-old fish could be reduced to nearly 1 / 2 to 1 / 3, even without medication. While this method does not allow for the shipment of 2-year-old fish without medication, it represents a significant effect considering that in areas with high infection rates, the percentage of 2-year-old fish that can be shipped sometimes falls below 70%.

[0052] In Example 2, similar to Example 1, doxycycline (antibiotic) and Ascophyllum nodosum extract were repeatedly administered to juvenile fish to enhance their immunity. Then, when the Edwardsiella species surviving in the bodies of 2-year-old fish began to become active (around June), a single dose of oxytetracycline (antibiotic) was administered. This was found to reduce the number of deaths due to Edwardsiella disease to less than 1 / 15 of that in Comparative Example 1. Furthermore, after the initial administration of oxytetracycline, the low mortality rate was maintained until shipment, and further administration was not necessary. This confirmed the effectiveness of oxytetracycline (antibiotic) in counteracting the decrease in immunity during winter and the increased activity of Edwardsiella species in early spring in farmed sea bream.

[0053] <Confirmation of synergistic effects of administering Ascophyllum nodosum extract (immunostimulant) and antibiotics against intracellular parasitic bacterial infections other than edwaziella disease in fish species other than red sea bream> (1) Nocardiosis in yellowtail A laboratory tank test was conducted to confirm the synergistic effect of administering Ascophyllum nodosum extract and antibiotics against nocardiosis in yellowtail.

[0054] (Control group 4) Ten yellowtail (normal fish) with an average weight of about 400g were inoculated with Nocardia bacteria in a 1-ton tank, and the survival rate after Nocardia infection was checked.

[0055] (Test Group 4) Ten yellowtail (normal fish) with an average weight of approximately 400g were inoculated with Nocardia bacteria in a 1-ton tank. From the 4th day after infection with Nocardia bacteria, they were treated with oxytetracycline (OTC) at a dose of 50mg per kg of body weight as an antibiotic for 5 days.

[0056] (Test Group 5) Treatment was carried out in the same manner as in Test Group 4, except that 4 mg of Ascophyllum nodosum extract per kg of body weight was orally administered as an immunostimulant for 5 days before inoculation with Nocardia bacteria and for 6 days from the 3rd day after inoculation with Nocardia bacteria.

[0057] Figure 5 shows the changes in the survival rate of yellowtail amberjack due to nocardiosis in control group 4 and experimental groups 4 and 5. A comparison between control group 4 and test groups 4 and 5 showed that monotherapy with over-the-counter (OTC) drugs did not have a significant therapeutic effect on nocardiosis. However, a significant difference in survival rate was observed when Ascophyllum nodosum extract was used in combination with OTC drugs. This suggests that for nocardiosis, enhancing immunity with Ascophyllum nodosum extract allows for more effective use of antibiotics, and that the synergistic effect of the immunostimulant and antibiotics minimizes bacterial carryover after antibiotic treatment.

[0058] (2) Mycobacterium infection in yellowtail Mycobacterial infection, like nocardiosis, is caused by intracellular parasitic bacteria that are a problem in farmed yellowtail. It is known that antibiotic administration alone is ineffective, and there is no effective treatment method. Therefore, in order to confirm the synergistic effect of administering Ascophyllum nodosum extract and antibiotics against mycobacterial infection in yellowtail, we conducted a tank test in the laboratory.

[0059] (Control group 5) Seventeen yellowtail (normal fish) with an average weight of approximately 250g were inoculated with Mycobacterium bacteria in a 1-ton tank, and the survival rate after Mycobacterium infection was checked.

[0060] (Test Group 6) Seventeen yellowtail (normal fish) with an average weight of approximately 250g were inoculated with Mycobacterium bacteria in a 1-ton tank. From the fourth day after Mycobacterium infection for 10 days, the fish were treated with 15mg of ST combination drug (an antibacterial agent containing sulfamethoxazole (SMX) and trimethoprim (TMP) in a 5:1 ratio) per kg of body weight.

[0061] (Test Group 7) Treatment was carried out in the same manner as in Test Group 6, except that 4 mg of Ascophyllum nodosum extract per kg of body weight was orally administered as an immunostimulant for 5 days before inoculation with Mycobacterium bacteria and for 5 days from the 3rd day after inoculation with Mycobacterium bacteria.

[0062] Figure 6 shows the changes in the survival rate of yellowtail amberjack due to mycobacterium disease in control group 5 and experimental groups 6 and 7. Comparison with control group 5 and test groups 6 and 7 showed that monotherapy with trimethoprim-sulfamethoxazole (ST) alone did not show much therapeutic effect against mycobacterial infections. However, a significant difference in survival rate was observed when ascophyllum nodosum extract and ST were used in combination. From this, it is thought that by enhancing immunity with ascophyllum nodosum extract, antibiotics can be used more effectively against mycobacterial infections, and it is presumed that the synergistic effect of the immunostimulant and antibiotic minimizes bacterial carryover after antibiotic treatment.

[0063] <Confirmation of synergistic effects of immunostimulants other than Ascophyllum nodosum extract and antibiotic administration against intracellular parasitic bacterial infections> In the treatment of nocardiosis in yellowtail, lactic acid bacteria were used as an immunostimulant instead of Ascophyllum nodosum extract, and a synergistic effect was confirmed when used in combination with antibiotics.

[0064] (Control group 6) Seventeen yellowtail (normal fish) with an average weight of approximately 250g were inoculated with Nocardia bacteria in a 1-ton tank, and the survival rate after Nocardia infection was confirmed.

[0065] (Test Group 8) Seventeen yellowtail (normal fish) with an average weight of approximately 250g were inoculated with Nocardia bacteria in a 1-ton tank. From the 4th day after infection with Nocardia bacteria, they were treated with oxytetracycline (OTC) at a dose of 50mg per kg of body weight as an antibiotic for 5 days.

[0066] (Test Group 9) Treatment was carried out in the same manner as in Test Group 8, except that 2 mg of lactic acid bacteria per kg of body weight was orally administered as an immunostimulant for 15 days before inoculation with Nocardia bacteria and for 5 days from the 3rd day after inoculation with Nocardia bacteria.

[0067] (Test Group 10) Treatment was carried out in the same manner as in Test Group 9, except that 4 mg of lactic acid bacteria per kg of body weight was orally administered as an immunostimulant.

[0068] Figure 7 shows the changes in the survival rate of yellowtail amberjack due to nocardiosis in control group 6 and experimental groups 8-10. A comparison between control group 6 and test groups 8-10 showed that monotherapy with over-the-counter (OTC) drugs had little therapeutic effect on nocardiosis, but a significant difference in survival rates was observed when lactic acid bacteria and OTC drugs were used in combination. While a direct comparison between Ascophyllum nodosum extract and lactic acid bacteria is difficult due to differences in test systems, water temperature, and dosage, it is believed that enhancing immunity with lactic acid bacteria allows for more effective use of antibiotics. It is also hypothesized that combining lactic acid bacteria, another immunostimulant besides Ascophyllum nodosum extract, with antibiotics may minimize bacterial carryover after antibiotic treatment due to their synergistic effect.

[0069] Based on these results, the present invention establishes a method for controlling intracellular parasitic bacteria that were previously difficult to treat with inactivated vaccines and for which treatment with highly susceptible antibiotics yielded little effect. Specifically, by administering immunostimulants along with scheduled antibiotic administration, and repeating natural infection and treatment multiple times to allow farmed fish to acquire immunity, it becomes possible to maintain a high shipment rate of farmed fish without worrying about mortality rates. Furthermore, since medication will no longer be administered outside of June for two-year-old fish, it becomes possible to administer medication systematically without worrying about shipment restriction periods. In this way, by applying the principle of vaccines and allowing farmed fish to acquire resistance to infection through repeated natural infection and treatment, it is believed that it is possible to achieve the same effect as a live vaccine.

[0070] Although embodiments of the present invention have been described above, the present invention is not limited in any way to the configurations described in the embodiments described above, and includes other embodiments and modifications that can be considered within the scope of the matters described in the claims.

Claims

1. A method for controlling intracellular parasitic bacterial infections in farmed fish, characterized in that, without selecting whether farmed young fish are infected with the target intracellular parasitic bacteria, a period is provided in which the farmed young fish are allowed to be naturally infected with the intracellular parasitic bacteria without administering any medication, and a period is provided in which antibiotics are administered at predetermined intervals in conjunction with the administration of an immunostimulant, thereby allowing the farmed young fish to acquire immunity to the intracellular parasitic bacteria and suppressing the onset of intracellular parasitic bacterial infections in farmed two-year-old fish.

2. The method for controlling intracellular parasitic bacterial infections in farmed fish according to claim 1, characterized in that the interval between administrations of the antibiotic in combination with the immunostimulant is one week to three months, which is the period for allowing the farmed fish to be naturally infected with the intracellular parasitic bacteria.

3. The method for controlling intracellular parasitic bacterial infections in farmed fish according to claim 1, characterized in that the interval between administrations of the antibiotic in combination with the immunostimulant is 4 to 8 weeks, which is the period for allowing the farmed fish to be naturally infected with the intracellular parasitic bacteria.

4. The method for controlling intracellular parasitic bacterial infections in farmed fish according to claim 1, characterized in that the antibiotic is a tetracycline antibiotic.

5. The method for controlling intracellular parasitic bacterial infections in farmed fish according to claim 4, characterized in that the antibiotic is doxycycline.

6. The method for controlling intracellular parasitic bacterial infections in cultured fish according to claim 1, characterized in that the immunostimulant is an extract of Ascophyllum nodosum.

7. The method for controlling intracellular parasitic bacterial infections in farmed fish according to claim 1, characterized in that the immunostimulant contains one or more of lactic acid bacteria, ascophyllan, and β-glucan.

8. The method for controlling intracellular parasitic bacterial infections in farmed fish according to claim 1, characterized in that an antibiotic is administered once at the time when intracellular parasitic bacteria surviving in the body of the two-year-old farmed fish begin to become active again.

9. The method for controlling intracellular parasitic bacterial infections in farmed fish according to claim 8, characterized in that the antibiotic administered to the two-year-old farmed fish is doxycycline or oxytetracycline.

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