Method for controlling intracellular parasitic bacterial infection in cultured 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]

[0001] The present invention relates to a method for preventing intracellular parasitic bacterial infection in farmed fish, which can minimize the number of deaths of farmed fish due to infection with intracellular parasitic bacteria before shipping and the disposal of farmed fish at the time of shipping. [Background technology]

[0002] In Japan, sea bream (madai) ranks second only to yellowtail in terms of the volume of farmed fish caught, making red sea bream farming a crucial industry. However, the damage caused by fish diseases has been increasing in recent years. In particular, damages caused by Edwardsiellosis (also known as tarda disease) in farmed red sea bream amounted to 760 million yen in 2019 and 520 million yen in 2020 (Ministry of Agriculture, Forestry and Fisheries: Information on the Occurrence of Fish Diseases). In particular, in areas with high levels of Edwardsiellosis infection, the number of fish shipped falls to less than 50% of the number of juveniles introduced due to mortality during rearing and the disposal of infected fish at the time of shipment. Edwardsiellosis is an infectious disease in farmed fish caused by infection with the gram-negative bacillus Edwardsiella tarda, E. piscicida, and E. anguillarum, and occurs year-round regardless of fish age. Edwardsiella bacteria are intracellular parasitic bacteria that tend to become chronic, leading to a decrease in the shipping volume of farmed fish such as red sea bream and flounder. Until now, infectious disease control in farmed fish has often relied on vaccines, and inactivated vaccines have been put into practical use for many diseases and have proven highly effective. Therefore, (Patent Document 1) has been researching a vaccine using inactivated cells of a strain derived from Edwardsiella tarda. Other research being conducted on preventive and therapeutic agents and methods using herbal medicines for the prevention and treatment of Edwardsiella (Patent Document 2) and fish feed containing medium-chain fatty acids with antibacterial activity (Patent Document 3). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-50300 [Patent Document 2] Japanese Patent Application Publication No. 1-75425 [Patent Document 3] International Publication No. 2000 / 10558 Summary of the Invention [Problem to be solved by the invention]

[0004] Here, immunity refers to a defense mechanism against pathogens such as viruses or bacteria (a mechanism that protects the living organism from pathogens), and the vaccines currently used to combat infectious diseases in farmed fish apply the principle of this immune response. Explained in more detail, when farmed fish become infected with a pathogen such as a virus or bacteria, the immune cells in the body memorize information about the pathogen, increasing resistance to subsequent infections, and the immune response can protect the living organism from the pathogen. However, because Edwardsiella bacteria grow inside living cells, they cannot be controlled with inactivated vaccines. Experimental research has shown that they can only be controlled with live vaccines, but because red sea bream farming is often done in seawater, it is virtually impossible to use live vaccines. Another possible control method besides vaccines is the use of antibiotics. Fosfomycin (trade name Fosmicin) has been approved as a fisheries drug for treating Edwardsiellosis in Perciformes, but as mentioned above, serious damage is still occurring, and it is reasonable to assume that sufficient therapeutic effects have not been achieved. For these reasons, no therapeutic drugs or methods for Edwardsiellosis have yet been shown to be as effective as expected, and there is a strong demand for the development of an effective method of preventing (treating) Edwardsiellosis, which causes enormous economic losses, particularly in red sea bream aquaculture. This is an issue not limited to Edwardsiellosis, but is common to all intracellular parasitic bacterial infections. The present invention has been made in consideration of the above circumstances, and aims to provide a method for preventing intracellular parasitic bacterial infections in farmed fish, which maximizes the effectiveness of antibiotics by combining an immunostimulant with an antibiotic and administering it repeatedly, thereby minimizing the onset of intracellular parasitic bacterial infections with short-term administration. [Means for solving the problem]

[0005] In accordance with the above-mentioned objectives, the method for preventing intracellular parasitic bacterial infections in farmed fish of the present invention involves administering antibiotics to farmed one-year-old fish at predetermined intervals in addition to feeding them an immunostimulant, and repeating natural infection with the target intracellular parasitic bacteria and post-infection treatment multiple times, thereby allowing the farmed one-year-old fish to acquire immunity to the intracellular parasitic bacteria and suppressing the onset of intracellular parasitic bacterial infections in farmed two-year-old fish. Here, fish in the first year of rearing are defined as yearlings, and fish reared from January of the following year onwards are defined as two-year-olds. Briefly, the theory behind the method for preventing intracellular parasitic bacterial infections in farmed fish according to the present invention applies the aforementioned vaccine principle, which involves intentionally setting aside periods during which farmed fish are infected with intracellular parasitic bacteria and then alternately administering medication to increase the farmed fish's resistance to infection. It is well-known from experience that administering antibiotics alone after an infection has occurred is insufficient to eradicate all bacteria already multiplying in the body, and that some bacteria are carried over into the farmed fish's bodies. Therefore, to minimize bacterial carryover, it is important to administer an immunostimulant and an antibiotic in combination during medication. To date, there have been no reports of improved performance (farming yield) in farmed fish using a combination of an immunostimulant and an antibiotic. Immunostimulants and antibiotics are administered to all one-year-old farmed fish during aquaculture, and there is no need to select farmed fish that have developed intracellular parasitic bacterial infections (infection with intracellular parasitic bacteria) at the time of administration. The immunostimulant is preferably administered continuously (daily) to farmed yearling fish during the period when they are infected with the target intracellular parasitic bacteria (the period when the target intracellular parasitic bacteria are thought to be active). The administration interval of the antibiotic is selected appropriately.

[0006] In the method for preventing intracellular parasitic bacterial infections in farmed fish of the present invention, the administration interval 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 during which the farmed one-year-old fish are naturally infected with the intracellular parasitic bacteria. Here, it is preferable to carry out (repeated) multiple cycles of administration, with a single administration period of 3 to 15 days (more preferably 5 to 10 days) and an administration interval of 1 week to 3 months (more preferably 4 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 an Ascophyllum nodosum extract. Ascophyllum nodosum is a large brown algae that grows in the coastal areas of Northern Europe and is used as livestock feed and a plant growth promoter. It 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. It is similar to fucoidan in that it contains L-fucose and sulfate groups, but differs in the composition of sugar residues. It is known to have apoptosis-inducing properties and antitumor activity against certain cancer cells. Preventive and therapeutic compositions using Ascophyllum nodosum extracts can contain Ascophyllum nodosum itself in any form, such as a dried powder, or can contain one or more compounds isolated from Ascophyllum nodosum. Any known method can be used to isolate the compounds contained in Ascophyllum nodosum, with solvent extraction being a preferred method. Ascophyllum nodosum, the source of the extract, can be sourced from any region. Because raw algae have a high water content, it is preferable to wash the algae with water to remove salt, then dry and powder the resulting extract. The average composition of the dried Ascophyllum nodosum powder used as the source of the extract is 5-10% protein, 45-60% carbohydrates, 17-20% ash, 2-4% lipids, and 10-12% water. The dried Ascophyllum nodosum powder may be pretreated with an aqueous acid solution to remove acid-soluble components. Extraction of compounds from Ascophyllum nodosum is performed using, for example, 10–100 times the mass of Ascophyllum nodosum dried powder in water. When Ascophyllum nodosum dried powder is added to water and stirred at room temperature (approximately 15–25°C) for 12–24 hours, ascophyllan is extracted into the water along with alginic acid and other components. If necessary, additional water can be added and the extraction continued for an additional 12–24 hours at approximately 100°C. The crude extract thus obtained contains other polysaccharides, proteins, carbohydrates, lipids, ash, minerals, pigments, polyphenols, and other components. Since these components are harmless to humans and animals, Ascophyllum nodosum extract can be used directly without separation when added to food or feed.

[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 preventing intracellular parasitic bacterial infections in farmed fish of the present invention, it is preferable to administer antibiotics once when the intracellular parasitic bacteria surviving in the bodies 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 order Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeformes, Salmoniformes or Anguilliformes.

[0014] The method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention is preferably applied to Edwardsiellosis in red sea bream, Edwardsiellosis in flounder, nocardiosis in Seriola of the order Perciformes, mycobacteriosis in Seriola of the order Perciformes, and paracolo disease (Edwardsiellosis) in eels. [Effects of the Invention]

[0015] The method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention involves repeatedly administering an immunostimulant and an antibiotic in combination during the period when the farmed fish are yearlings of one year, thereby enhancing immunity through a synergistic effect, minimizing the number of deaths among yearlings of one year, and further minimizing the onset of intracellular parasitic bacterial infections in two-year-old fish before shipping. Because all fish have immune systems, the above method can be applied to all fish species.

[0016] In the method of the present invention for preventing intracellular parasitic bacterial infections in farmed fish, if the interval between administration of antibiotics in combination with an immunostimulant is 1 week to 3 months (more preferably 4 to 8 weeks) as the period during which farmed yearling fish are allowed to become naturally infected with intracellular parasitic bacteria, the effectiveness of the antibiotics can be effectively utilized to maximize the effect and the dosage can be reduced.

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

[0018] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, when the antibiotic is doxycycline, it has high intracellular permeability and is highly effective in treating intracellular parasitic bacteria that grow within the cells.

[0019] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, when the immunostimulant is an Ascophyllum nodosum extract, the number of intracellular parasitic bacteria can be minimized through a synergistic effect with the antibiotic, thereby reducing the mortality of yearling fish, preventing the recurrence of intracellular parasitic bacterial infections, and preventing the carryover of intracellular parasitic bacteria from yearling fish to two-year-old fish.

[0020] In the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention, when the immunostimulant contains one or more of lactic acid bacteria, ascophyllan, and β-glucan, the number of intracellular parasitic bacteria is minimized through a synergistic effect with the antibiotic, thereby reducing the mortality of yearling fish, preventing recurrence of intracellular parasitic bacterial infections, and preventing carryover of intracellular parasitic bacteria from yearling fish to two-year-old fish.

[0021] In the method for preventing intracellular parasitic bacterial infections in farmed fish of the present invention, if antibiotics are administered once at the time when intracellular parasitic bacteria surviving in the bodies of two-year-old farmed fish begin 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 farmed fish according to the present invention, when the antibiotic administered to two-year-old farmed fish is doxycycline or oxytetracycline, a low mortality rate is maintained until shipping.

[0023] In the method for preventing intracellular parasitic bacterial infections in farmed fish of the present invention, when the farmed fish belong to the order Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeidae, Salmoniformes or Anguilliformes, various farmed fish can be protected from infections caused by intracellular parasitic bacteria.

[0024] When the method for controlling intracellular parasitic bacterial infections in farmed fish according to the present invention is applied to Edwardsiellosis in red sea bream, Edwardsiellosis in flounder, nocardiosis in Seriola of the order Perciformes, mycobacteriosis in the order Perciformes, and paracolovirus in eels, it can reduce damage to the entire aquaculture industry, regardless of the species of farmed fish. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows a protocol for a test to confirm the therapeutic efficacy of antibiotics used to treat Edwardsiellosis in farmed red sea bream. [Figure 2] FIG. 1 shows the results of a test to confirm the therapeutic effect of antibiotics used to treat Edwardsiellosis in farmed red sea bream. [Figure 3] FIG. 1 shows the results of a test to confirm the immune acquisition effect of an immunostimulant used in the treatment of Edwardsiellosis in farmed red sea bream. [Figure 4] FIG. 1 shows the protocol for a test to confirm the optimal medication method for treating Edwardsiellosis in farmed red sea bream. [Figure 5] FIG. 1 shows the results of a test to confirm the synergistic effect of administering an Ascophyllum nodosum extract and an antibiotic against nocardiosis in yellowtail. [Figure 6] FIG. 1 shows the results of a test to confirm the synergistic effect of administering an Ascophyllum nodosum extract and an antibiotic against mycobacteriosis in yellowtail. [Figure 7] FIG. 1 shows the results of a test to confirm the synergistic effect of administering lactic acid bacteria and antibiotics against nocardiosis in yellowtail. DETAILED DESCRIPTION OF THE INVENTION

[0026] Next, with reference to the accompanying drawings, embodiments embodying the present invention will be described to aid in understanding the present invention. One embodiment of the present invention relates to a method for preventing intracellular parasitic bacterial infections in farmed fish. This involves repeating natural infection and treatment while using an immunostimulant and an antibiotic in combination at the age of one year, thereby increasing the acquired immunity to the target bacteria and minimizing the residual bacteria in the fish's body after medication, thereby preventing the recurrence of intracellular parasitic bacterial infections such as Edwardsiellosis, and reducing mortality and waste at the time of shipping in two-year-old fish without the need for medication.

[0027] Previous fish disease countermeasures, including Edwardsiellosis, involved identifying the cause and initiating medication once mortality was confirmed. However, this medication method often involved delays in administering medication, and although mortality initially decreased after the medication stopped, the disease would recur after the medication's effectiveness wore off, necessitating repeated medication. This suggests that even if mortality was reduced to zero after medication, there is a high possibility that intracellular parasitic bacteria remain in the body. As a result, increased medication dosage led to increased production costs and an increase in cumulative mortality, placing a strain on production sites. In particular, with regard to Edwardsiellosis in farmed red sea bream, the deaths of two-year-old fish have become a problem, but it is thought that the actual infection occurs in year-old fish. This is because even if year-old fish are infected with Edwardsiellosis, the activity of the Edwardsiellosis bacteria temporarily ceases during the winter, so the number of deaths among year-old fish does not increase significantly, and it appears that the infection has ceased. However, in reality, the Edwardsiellosis bacteria are carried over, and then, in early spring, the activity of the Edwardsiellosis bacteria becomes active, causing Edwardsiellosis to develop in two-year-old fish.

[0028] Therefore, in order to prevent the onset of Edwardsiellosis in two-year-old fish, measures must be taken while the fish are still one year old. Specifically, it is thought that it is necessary to increase the immunity of one-year-old farmed red sea bream to Edwardsiellosis by administering immunostimulants and antibiotics at regular intervals to the fish during aquaculture, and repeating natural infection with Edwardsiellosis and post-infection treatment multiple times. Here, the administration interval of the immunostimulant and the antibiotic is preferably, but not limited to, one week to three months (more preferably four to eight weeks). The single dose, number of doses, and total dose of each of the immunostimulant and the antibiotic are selected appropriately.

[0029] As antibiotics, tetracycline antibiotics are preferred, and doxycycline, which has high intracellular penetration and is particularly effective against intracellular parasitic bacteria, is preferably used, but is not limited to these. For example, drugs that have previously been considered ineffective in aquaculture despite being drug-sensitive, have only failed to provide therapeutic benefit because they were administered at the late stage of infection. However, if administered early in the infection process, there is a high possibility that they will be effective. Furthermore, if medication is administered with an immune-centered approach, the options for available drugs will be greatly expanded, and the problem of drug-resistant bacteria can be overcome. Specifically, oxolinic acid, sulfonamides in general, florfenicol, fosfomycin, etc., can be used. For example, in the case of Edwardsiellosis in farmed sea bream, doxycycline should be administered for 3 to 15 days (preferably 5 to 10 days) per treatment, preferably multiple times. Furthermore, it is desirable to administer 20 to 400 mg / kg BW (preferably 40 to 80 mg / kg BW) per day. The immunostimulant is preferably, but not limited to, an Ascophyllum nodosum extract. For example, an immunostimulant made from beta-glucan or lactic acid bacteria may also be used.

[0030] As described above, if we repeatedly administer immunostimulants and antibiotics to farmed red sea bream that are one year old, and they acquire immunity while they are one year old, it is thought that the onset of Edwardsiellosis can be effectively suppressed even when they become two years old. However, from winter in yearlings to around May in two-year-old fish, the activity of Edwardsiella bacteria remaining in the body weakens, and immune function also declines. Therefore, if no preventative measures are taken when Edwardsiella bacteria begin to become active in early spring, two-year-old fish may develop Edwardsiellosis before shipping. Therefore, administering an antibiotic to two-year-old fish once when Edwardsiella bacteria begin to become active (around April to July) can more reliably prevent the onset of Edwardsiellosis. The antibiotic administered at this time can be either doxycycline or oxytetracycline, and from a cost perspective, oxytetracycline, which is cheaper than doxycycline, is preferable, but is not limited to these.

[0031] In this embodiment, the explanation has been focused on Edwardsiellosis in farmed red sea bream, but because all fish have immune systems, 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 Edwardsiellosis in flounder, nocardiosis in the genus Seriola (Perciformes), mycobacteriosis in the genus Seriola (Perciformes), and paracolo disease (Edwardsiellosis) in eels, which are all caused by the same intracellular parasitic bacteria. [Example]

[0032] Examples carried out to confirm the effects of the present invention will be described below, but these examples are not intended to limit the technical scope of the present invention in any way.

[0033] <Confirmation test of antibiotic treatment effect> A study was conducted to verify the therapeutic (prophylactic) effect of antibiotics selected as treatments for Edwardsiellosis in farmed red sea bream. The study protocol is shown in Figure 1.

[0034] (Test area 1) After deaths due to Edwardsiellosis were confirmed in approximately 5,000 two-year-old farmed red sea bream (average weight 1 kg) in a farming raft (8 m x 8 m x 5 m), the antibiotic doxycycline was administered orally (feeding with a spreading agent). The dosage was 80 mg per kg of body weight, and the number of doses was 5 consecutive days, with a 14-day rest period (dose interval) between doses to ensure safety. A total of 3 doses were administered.

[0035] (Test area 2) The same procedures as in Test Group 1 were followed, except that the single dose was 40 mg per kg of body weight and the number of doses was 10 consecutive days.

[0036] (Control areas 1 and 2) Control areas 1 and 2 (one aquaculture raft each) were treated in the same manner as test area 1, except that no medication was administered.

[0037] Figure 2 shows the changes in the cumulative number of deaths due to Edwardsiellosis in test plots 1 and 2 and control plots 1 and 2. A comparison of test areas 1 and 2 with control areas 1 and 2 confirmed the therapeutic effect of doxycycline (antibiotic) against Edwardsiellosis. Furthermore, a comparison of test plots 1 and 2 showed that although the total dosage of doxycycline was the same, the cumulative number of deaths in test plot 2 was lower than in test plot 1. This indicates that administering a low concentration of doxycycline over a long period of time, as in test plot 2, provides a greater therapeutic effect than administering a high concentration of doxycycline over a short period of time, as in test plot 1. However, even after repeated administration of doxycycline, the number of deaths never reached zero, and the cumulative number of deaths continued to increase, confirming that starting doxycycline administration to fish at two years of age is not cost-effective.

[0038] <Confirmation of therapeutic effects of immunostimulants> A test was conducted to confirm the immune effect of an immunostimulant against Edwardsiellosis in farmed red sea bream.

[0039] (Test area 3) Approximately 10,000 young-of-the-year farmed red sea bream (average weight 150g) were treated with the antibiotic doxycycline and the immunostimulant Ascophyllum nodosum extract in aquaculture rafts (8m x 8m x 5m). 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. 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 area 3) Control group 3 was treated in the same manner as test group 3, except that no Ascophyllum nodosum extract was administered.

[0041] The changes in the number of deaths due to Edwardsiellosis in test area 3 and control area 3 are shown in Figure 3. Comparison of test group 3 and control group 3 revealed a synergistic effect of the combined use of Ascophyllum nodosum extract (immunostimulant) and antibiotics against Edwardsiellosis. While bacteriostatic antibiotics are highly effective at stopping the movement of bacteria, they have little bactericidal effect when used alone. However, when used in combination with an immunostimulant, the bactericidal effect is thought to be enhanced by collaboration with the immune system. Therefore, the combined use of Ascophyllum nodosum extract not only reduces the number of deaths, but is also thought to have a synergistic effect in reducing the number of Edwardsiella bacteria in the body, and is expected to prevent carryover of Edwardsiella bacteria from one-year-old fish to two-year-old fish.

[0042] For the farmed red sea bream in test area 3 and control area 3, a portion was fed in February when they were two years old (test area 3: 4,620 fish, control area 3: 4,340 fish), and the cumulative number of deaths up to the time of shipping was then checked. As shown in the bottom of Figure 3, the final yield from feeding to shipping (number of fish at shipping / number of fish at feeding x 100) was 85.8% in control area 3, but 93.3% in test area 3. This suggests that administering doxycycline alone to yearling fish has limited effectiveness in preventing the onset of the disease in two-year-old fish, but that combining it with an immunostimulant produces a continuous preventive effect in two-year-old fish, leading to improved performance (yield). Furthermore, the yield, which is usually around 70%, increased to around 85%-93% due to the acquisition of resistance to infection in one-year-old fish, proving that the acquisition of resistance to infection in one-year-old fish is important.

[0043] <Confirmation of lifetime medication schedule> Based on the results of the above tests, a test was conducted to confirm the optimal medication method for farmed red sea bream from introduction to shipment. Approximately 10,000 red sea bream were farmed in each farming raft (fish pen) (8m x 8m x 5m), and the average weight of the yearling farmed red sea bream at the start of farming was 30g. As with the above test, medication was administered on a farming raft basis, and the test period was approximately two years. The test protocol is shown in Figure 4.

[0044] (Comparative Example 1: No medication) Three farming rafts (net cages) were used to raise 10,000 farmed red sea bream without medication from the start of farming in June until February when the fish reached their second year of age (their first-year age). Five thousand farmed red sea bream were then randomly selected from each of these rafts and divided into two net cages, where they were farmed without medication until October of the following year. The cumulative number of deaths from Edwardsiellosis from July to September, when deaths from Edwardsiellosis at two years of age increase, was then investigated.

[0045] (Comparative Example 2: Normal (conventional) measures) From the three culture rafts (net cages) used in Comparative Example 1, which were reared without medication from the start of culture in June until February when the fish reached two years of age, 5,000 cultured red sea bream were randomly selected from each of the two cages and reared without medication until death due to Edwardsiellosis was confirmed around June when the fish reached two years of age. From July onward, three cycles of doxycycline were administered, each at a dose of 40 mg per kg of body weight for 10 days. Since no reduction in mortality was observed during this period, a five-day withdrawal period (interval between doses) was used. This withdrawal period was not intended to confer immunity to the fish, but rather follows the current common practice of administering medication only after deaths were confirmed.

[0046] Example 1 Five culture rafts (net pens) containing 10,000 young-of-the-year-old red sea bream were each administered 4 mg of Ascophyllum nodosum extract per kg of body weight daily (approximately 150 times) from the start of culture on June 15 through November 30 to confirm the synergistic effects of the medication. Starting in July, three cycles of doxycycline were administered, each at a dose of 40 mg per kg of body weight for 10 days. A 45-day rest period (interval between doses) was then allowed to allow for natural infection with Edwardsiella spp. Five thousand two-year-old red sea bream were randomly selected from each of the culture rafts (net pens) and then cultured without medication.

[0047] Example 2 During the yearling period, 5,000 farmed red sea bream were randomly selected from the same five farming rafts (cages) as in Example 1 and grown to two years of age. These were then distributed to five farming rafts (cages). In June (when Edwardsiella begins to become active), the antibiotic oxytetracycline was administered once. Therefore, Example 2 differs from Example 1 in that the antibiotic oxytetracycline was administered once in June when the fish were two years old.

[0048] The cumulative number of deaths due to Edwardsiellosis in 2-year-old fish in Comparative Examples 1 and 2 and Examples 1 and 2 is shown in Table 1.

[0049] [Table 1]

[0050] As can be seen from Table 1, in Comparison Example 1, in which no measures against Edwardsiellosis were taken from the start of farming until shipment (farming 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 Edwardsiellosis in 2-year-old fish. In Comparative Example 2, the number of deaths was reduced to about half of that in Comparative Example 1. However, as with conventional fish disease countermeasures, it was found that administering doxycycline (antibiotic) after the onset of the disease was not sufficiently effective for large 2-year-old fish.

[0051] In Example 1, it was found that by feeding Ascophyllum nodosum extract daily to yearlings to boost their immunity while also administering doxycycline (an antibiotic), the incidence of Edwardsiellosis in two-year-old fish could be reduced by approximately one-half to one-third, even without medication. This method is not enough to allow fish to be shipped without medication at two years of age, but considering that in heavily infected areas, the percentage of two-year-old fish eligible for shipment can be less than 70%, it can be said to be a significant effect.

[0052] In Example 2, as in Example 1, doxycycline (antibiotic) and Ascophyllum nodosum extract were repeatedly administered to yearlings to enhance their immunity, and then oxytetracycline (antibiotic) was administered once to two-year-old fish around June, when the Edwardsiella bacteria living in their bodies begin to become active. This demonstrated that the number of deaths due to Edwardsiella disease could be reduced to 1 / 15 or less of that in Comparative Example 1. Furthermore, after the first administration of oxytetracycline, the low mortality rate was maintained until shipping, and further administration was not necessary. This confirmed the effectiveness of oxytetracycline (antibiotic) administration against the weakening of immunity in farmed red sea bream during the winter and the increased activity of Edwardsiella bacteria in early spring.

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

[0054] (Control group 4) Nocardia bacteria were inoculated into 10 yellowtail (normal fish) with an average weight of approximately 400g in a 1 ton tank, and the survival rate after infection with Nocardia bacteria was confirmed.

[0055] (Test area 4) Ten yellowtail (normal fish) with an average weight of approximately 400 g were inoculated with Nocardia bacteria in a 1 ton tank, and treated with 50 mg of oxytetracycline (OTC) per kg of body weight as an antibiotic for five days starting from the fourth day after Nocardia infection.

[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 and for 6 days from the 3rd day after inoculation with Nocardia.

[0057] The changes in the survival rate of yellowtail due to nocardiosis in control area 4 and experimental areas 4 and 5 are shown in Figure 5. A comparison of control group 4 with test groups 4 and 5 showed that administration of OTC alone was not very effective against nocardiosis, but a significant difference in survival rate was observed when Ascophyllum nodosum extract was used in combination with OTC. This suggests that antibiotics can be used more effectively against nocardiosis by boosting immunity with Ascophyllum nodosum extract, and that the synergistic effect of the immunostimulant and antibiotics minimizes bacterial carryover after antibiotic treatment.

[0058] (2) Mycobacterial disease in yellowtail Like nocardiosis, mycobacteriosis is an intracellular parasitic bacterium that is a problem in farmed yellowtail. It is known that administering antibiotics alone has little effect on treatment, and there is no effective treatment method. Therefore, a laboratory tank test was conducted to confirm the synergistic effect of Ascophyllum nodosum extract and antibiotics on mycobacteriosis in yellowtail.

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

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

[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 mycobacteria and for 5 days from the 3rd day after inoculation with mycobacteria.

[0062] Figure 6 shows the changes in survival rate of yellowtail due to mycobacterial disease in control area 5 and experimental areas 6 and 7. Comparison of control group 5 with experimental groups 6 and 7 showed that administration of the ST combination alone was not very effective against mycobacteriosis, but a significant difference in survival rate was observed when Ascophyllum nodosum extract was used in combination with the ST combination. This suggests that antibiotics can be used more effectively against mycobacteriosis by boosting immunity with Ascophyllum nodosum extract, and that the synergistic effect of the immunostimulant and antibiotics minimizes bacterial carryover after antibiotic treatment.

[0063] <Confirmation of synergistic effects of immunostimulants other than Ascophyllum nodosum extract and antibiotics against intracellular bacterial infections> In the control 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 250 g were inoculated with Nocardia bacteria in a 1 ton tank, and the survival rate after infection with Nocardia bacteria was confirmed.

[0065] (Test area 8) Seventeen normal yellowtail (yellowtail) fish weighing an average of 250 g were inoculated with Nocardia bacteria in a 1 ton tank, and treated with 50 mg of oxytetracycline (OTC) per kg of body weight as an antibiotic for five days from the fourth day after Nocardia infection.

[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] The changes in the survival rate of yellowtail due to nocardiosis in control area 6 and experimental areas 8 to 10 are shown in Figure 7. A comparison of control area 6 and test areas 8 to 10 showed that administration of OTC alone had almost no therapeutic effect against nocardiosis, but a significant difference in survival rate was observed when lactic acid bacteria and OTC were used in combination. As the test system, water temperature, dosage, etc. differ between Ascophyllum nodosum extract and lactic acid bacteria, a general comparison cannot be made; however, it is thought that by boosting immunity with lactic acid bacteria, antibiotics can be used more effectively, and it is also speculated that by combining antibiotics with lactic acid bacteria, which are immunostimulants other than Ascophyllum nodosum extract, the synergistic effect can minimize bacterial carryover after antibiotic treatment.

[0069] Based on these results, the present invention establishes a method for controlling intracellular parasitic bacteria, which have traditionally been difficult to treat with inactivated vaccines and for which treatment with highly susceptible antibiotics has proven ineffective. Specifically, by administering scheduled antibiotics along with the feeding of immunostimulants and repeatedly repeating natural infection and treatment to cultivate cultured fish, it becomes possible to maintain a high shipping rate for cultured fish without worrying about mortality. Furthermore, since treatment is no longer required outside of June for two-year-old fish, planned administration is possible without worrying about shipping restrictions. Thus, by applying the principles of vaccination and cultivating resistance in cultured fish through repeated natural infection and treatment, it is believed possible to achieve the same effectiveness as a live vaccine.

[0070] The above describes an embodiment of the present invention, but the present invention is not limited to the configurations described in the above embodiment, and also includes other embodiments and modifications that are possible within the scope of the matters described in the claims.

Claims

1. A method for preventing intracellular parasitic bacterial infections in farmed fish, characterized by administering antibiotics to farmed one-year-old fish at predetermined intervals in addition to feeding them an immunostimulant, and repeating natural infection with the target intracellular parasitic bacteria and post-infection treatment multiple times, thereby causing the farmed one-year-old 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. A method for preventing intracellular parasitic bacterial infections in farmed fish as described in claim 1, characterized in that the administration interval of the antibiotic in combination with the immunostimulant is 1 week to 3 months, which is the period during which the farmed yearling fish are allowed to naturally become infected with the intracellular parasitic bacteria.

3. The method for preventing intracellular parasitic bacterial infections in farmed fish described in claim 1, characterized in that the administration interval of the antibiotic in combination with the immunostimulant is 4 to 8 weeks, which is the period during which the farmed yearling fish are naturally infected with the intracellular parasitic bacteria.

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

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

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

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

8. A method for preventing intracellular parasitic bacterial infections in farmed fish as described in claim 1, characterized in that antibiotics are administered once when the intracellular parasitic bacteria living in the bodies of the two-year-old farmed fish begin to become active again.

9. 8. The method for controlling intracellular parasitic bacterial infections in farmed fish according to claim 7, wherein the antibiotic administered to the two-year-old farmed fish is doxycycline or oxytetracycline.

10. 2. The method for controlling intracellular parasitic bacterial infections in farmed fish according to claim 1, wherein the farmed fish belong to the order Perciformes, Pleuronectiformes, Tetraodontiformes, Clupeformes, Salmoniformes or Anguilliformes.

11. The method for controlling intracellular parasitic bacterial infections in farmed fish according to claim 1, characterized in that it is applicable to Edwardsiellosis in red sea bream, Edwardsiellosis in flounder, nocardiosis in Seriola of the order Perciformes, mycobacteriosis in the genus Seriola of the order Perciformes, and paracolopathia in eels.

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