JP2025029653A - Preventive and therapeutic agents for infectious diseases in fish - Google Patents

Abstract

The present invention provides a safe and simple agent for preventing and treating infectious diseases in fish that does not rely on antibiotics or the like.
The present invention relates to a preventive or therapeutic agent for infectious diseases in fish, which contains sucrose and/or 1-kestose as active ingredients.
[Selected Figure] Figure 1

Description

The present invention relates to an agent for preventing or treating infectious diseases in fish.

In recent years, the demand for marine products has been expanding worldwide, but marine resources are limited, and with no prospect of an increase in production from fishing boats, a shift to aquaculture is underway. However, in aquaculture, the occurrence of fish diseases is unavoidable due to high-density farming, and fish diseases are causing a lot of damage to commercial fish such as yellowtail, pufferfish, and eels, with the greatest damage occurring to yellowtail at approximately 4 billion yen, and overall losses amounting to approximately 10 to 11 billion yen per year. Therefore, measures to combat fish diseases are considered to be an important issue for the sustainable development of aquaculture.

Diseases that cause great damage to farmed fish are mainly viral and bacterial infections, but most of them are bacterial infections. For example, Edwardsiella disease is an infectious disease caused by Edwardsiella bacteria, and infects marine fish such as flounder and red sea bream as well as eels, resulting in great economic losses. In eels, it is called paracolo disease, and can affect everything from glass eels to adult eels. When it develops, redness and enlargement of the anus, redness and swelling of the anterior abdomen are observed, and numerous abscesses and ulcers are formed in the liver, kidneys, etc. Cold water disease is a disease caused by Flavobacterium psychrophilum, which infects freshwater fish such as sweetfish, rainbow trout, and carp, and is characterized by lesions such as erosions and ulcers that appear on the caudal peduncle. In eels, a disease called columnaris disease caused by a similar flavobacterium, Flavobacterium columnare, has been reported.

Antibiotics are used to treat and prevent such bacterial infections. However, there are concerns that the use of antibiotics can lead to the emergence of drug-resistant bacteria and that antibiotics may remain in the bodies of fish, resulting in safety issues. Meanwhile, vaccines are being developed, but the time and cost required for administration is significant, hindering their widespread use. For this reason, there is a demand for the establishment of safe and easy methods for preventing and treating infectious diseases that do not rely on antibiotics.

In response to this, a method for preventing and treating infectious diseases has been proposed in which lactose fructose oligosaccharides or soybean oligosaccharides are administered to fish (Patent Documents 1 and 2). However, there is no specific disclosure regarding the preventive and therapeutic effects against the above-mentioned bacterial infectious diseases.

Japanese Patent Application Publication No. 10-45605 JP 2022-120840 A

Yaqiu Liu et al.,Front.Microbiol.,09 August 2022 Vol.13 Yibin Yang et al., Front.Immunol.,03 June 2022 Vol.13 Jiamin Li et al., Front.Nutr.19 October 2022 Vol9 Hao-Jun Zhu et al., MicrobiologyOpen Vol.9,Isuue 5 2020 Chunling Wang et al., Fish & Shellfish Immunology Vol.106,November 2020 Xiaolin Ye et al., Frontieres in Endocrinology, December 2021 vol.12 Sytze de Roock et al., Clinical Nutrition, December 2011 vol.30 Yi Liu et al., Multiple Sclerosis and Related Disorders Vol. 72, April 2023 Sage J B Dunham et al., mBio, 2022 Volume 13 Issue 6 Pagakrong Wanapaisan et al., Neurodegenerative Diseases, 2022;22(2):43-54. Honggang Yin et al., FEMS Microbiology Letters, Volume 370, 2023

The object of the present invention is to provide a safe and easy-to-use agent for preventing and treating infectious diseases in fish.

As a result of intensive research conducted by the inventors to solve the above problems, they discovered that feeding sucrose or 1-kestose to fish promotes the proliferation of Romboutsia bacteria, which are beneficial bacteria in the intestines, while suppressing the proliferation of pathogenic bacteria such as Edwardsiella bacteria and Flavobacterium bacteria, thereby making it possible to treat and prevent infections caused by these pathogenic bacteria, and thus completed the present invention.

That is, the present invention is a preventive or therapeutic agent for infectious diseases in fish that contains sucrose and/or 1-kestose as active ingredients.

The present invention also relates to a fish intestinal regulator that contains sucrose and/or 1-kestose as active ingredients.

The present invention also relates to an agent for inhibiting the proliferation of bacteria of the genus Romboutsia and bacteria of the genus Edwardsiella and/or Flavobacterium in the intestines of fish, which contains sucrose and/or 1-kestose as active ingredients.

The present invention also relates to a feed efficiency improver that contains sucrose and/or 1-kestose as active ingredients.

The present invention also relates to a method for preventing or treating an infectious disease, which comprises administering sucrose and/or 1-kestose to fish.

The present invention also relates to a method for improving the intestinal environment of fish, which comprises administering sucrose and/or 1-kestose to the fish.

The present invention allows the proliferation of Romboutsia bacteria, which are useful bacteria in the intestines, while suppressing the proliferation of pathogenic bacteria such as Edwardsiella bacteria and Flavobacterium bacteria, thereby making it possible to treat or prevent infectious diseases caused by these bacteria.

1 is a graph showing the relative occupancy rates of Romboutsia, Edwardsiella, and Flavobacterium bacteria in the eel intestinal bacterial flora in the test group and the control group in Example 1. 1 is a graph showing the concentrations of lactic acid and acetic acid in the intestines of eels in the test group and the control group in Example 1. 1 is a graph showing the growth rate of Edwardsiella bacteria in a test group and a control group in Example 4.

The target fish for the infectious disease preventive or therapeutic agent of the present invention (hereinafter referred to as "infectious disease preventive and therapeutic agent") are not particularly limited, but include, for example, freshwater fish such as eel, rainbow trout, sweetfish, tilapia, carp, crucian carp, and catfish, and saltwater fish such as flounder, red sea bream, yellowtail, striped jack, and coho salmon, and among these, it is preferably used for eel, rainbow trout, sweetfish, tilapia, carp, flounder, red sea bream, etc.

The active ingredients of the infectious disease prevention and treatment agent of the present invention are sucrose and/or 1-kestose. Among these, 1-kestose is preferred because of its superior effect of promoting the proliferation of bacteria of the genus Romboutsia and its superior effect of inhibiting the proliferation of pathogenic bacteria such as bacteria of the genus Edwardsiella and Flavobacterium.

The infectious diseases that can be treated with the infectious disease preventive and therapeutic agent of the present invention include, but are not limited to, bacterial infectious diseases such as Edwardsiella, eel paracolo disease and columnaris disease, cold water disease, furunculosis, alpha-hemolytic streptococcosis, vibriosis, nocardiosis, bacterial gill disease, gliding bacteriosis, and beta-hemolytic streptococcosis, and among these, the agent is preferably used for infectious diseases caused by infection with Edwardsiella bacteria and/or Flavobacterium bacteria. Infectious diseases caused by infection with Edwardsiella bacteria include Edwardsiella and eel paracolo disease, and infectious diseases caused by infection with Flavobacterium bacteria include cold water disease and eel columnaris disease.

The dosage of the infectious disease preventive and therapeutic agent of the present invention is not particularly limited and can be set appropriately depending on the type of fish to which it is applied, the type of infectious disease, symptoms, etc., but the addition rate of various oligosaccharides should be 0.1% to 10% of the fish body weight, and should be adjusted appropriately depending on the fish species and size. For example, the dosage can be about 1,280 mg/kg of fish body weight per day.

The method of application of the infectious disease preventive and therapeutic agent of the present invention is not particularly limited, but oral administration is preferred, and the active ingredient can be formulated as an oral preparation or added to fish feed and fed. The formulation form is not particularly limited, and any dosage form such as granules or tablets can be used, and can be formulated according to known preparation methods. Feed to which the infectious disease preventive and therapeutic agent of the present invention has been added can also be prepared according to conventional methods, and sucrose and/or 1-kestose can be added to raw feed such as fish meal, wheat flour, starch, calcium phosphate, etc., so that the amount of sucrose and/or 1-kestose is, for example, about 3.5 to 4.0% by mass of the total feed, and the feed can be made into any form such as moist pellets or dry pellets.

By administering the infectious disease preventive and therapeutic agent of the present invention to fish, the intestinal bacterial environment of the fish is improved, and an effect of preventing and treating infectious diseases is obtained. That is, many pathogenic bacteria of bacterial infectious diseases, such as Edwardsiella bacteria, which are the causative bacteria of Edwardsiella disease, and Flavobacterium bacteria, which are the causative bacteria of cold water disease, are facultative pathogenic bacteria that are normally present in environmental water and intestines. However, by administering the infectious disease preventive and therapeutic agent of the present invention, useful bacteria, Romboutsia bacteria, are proliferated in the intestines of fish (Non-Patent Documents 6-7), while the proliferation of pathogenic bacteria such as Edwardsiella bacteria and Flavobacterium bacteria is suppressed, making it possible to prevent or treat infectious diseases caused by these. The useful bacteria Romboutsia bacteria are widely known to exist as normal bacteria in the intestines of fish, and have been reported to exist as normal bacteria in eels, tilapia, herring, crucian carp, catfish, mint crab, etc. (Non-Patent Documents 1 to 5). Furthermore, administration of sucrose and/or 1-kestose promotes the growth of fish, thereby improving feed efficiency. Therefore, the present invention includes, in addition to an infectious disease preventive and therapeutic agent for fish containing sucrose and/or 1-kestose as an active ingredient, an intestinal regulator for fish, an agent for promoting the growth of Romboutsia bacteria in the intestines of fish, an inhibitor of the growth of Edwardsiella bacteria, an inhibitor of the growth of Flavobacterium bacteria, an agent for improving feed efficiency, and methods thereof.

The infectious disease preventive and therapeutic agent of the present invention may be in a form that is labeled for use in preventing and treating bacterial infectious diseases such as Edwardsiellosis, paracolo disease, and cold water disease, for intestinal regulation, for improving the intestinal environment, for inhibiting the proliferation of bacteria of the genus Romboutsia in the intestines, and for inhibiting the proliferation of bacteria of the genus Edwardsiella and/or Flavobacterium. For example, this may be indicated on the product itself, container, packaging, instructions, and package insert, or in advertisements such as pamphlets and posters, promotional materials, webinars, and SNS screens.

In addition, since Romboutsia bacteria are normally present in the intestines of various animals, animals can also be the subjects of administration, including mammals such as humans, dogs, cats, monkeys, chimpanzees, cows, horses, and sheep. In humans, Romboutsia bacteria are known to be involved in various diseases and physiological functions, and for example, the association between Romboutsia bacteria and diabetes, demyelinating optic neuritis, Alzheimer's, and improvement of vascular endothelial function has been reported (Non-Patent Documents 8 to 11). Therefore, the Romboutsia bacteria proliferation agent of the present invention containing sucrose and/or 1-kestose as an active ingredient can prevent or improve diseases such as diabetes, demyelinating optic neuritis, and Alzheimer's, as well as hypertension, dyslipidemia, obesity, metabolic syndrome, and arteriosclerosis associated with decreased vascular endothelial function.

The Romboutsia bacteria proliferation agent of the present invention can be formulated by combining the above-mentioned active ingredient with a pharma- ceutically acceptable carrier as necessary. Examples of pharma-ceutically acceptable carriers include glucose, lactose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, water, and physiological saline. Furthermore, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonicity agents, and excipients can be added as necessary. The dosage form is not particularly limited, but examples include liquids, powders, granules, capsules, and tablets, and can be prepared according to conventional methods. The dosage of the Romboutsia bacteria proliferation agent of the present invention is not particularly limited, but for example, the daily dosage for adults is about 0.1 to 5 g per kg of body weight, and preferably about 0.5 to 1 g.

The Romboutsia bacteria proliferation agent of the present invention can be in the form of food or drink, and is prepared by blending the above-mentioned active ingredient with known food additives and/or food materials in accordance with a conventional method, and the form is not particularly limited. Examples of the form include starch-based foods such as bread, biscuits, pancakes, noodles, and tablet candy, confectioneries such as gum, candy, and Japanese sweets, meat foods such as ham and sausage, fish foods such as chikuwa and kamaboko, seafood foods, seasonings such as dressing, soy sauce, jam, and furikake, beverages such as tea, juice, soft drinks, and alcoholic beverages, etc.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Example 1
Effect of 1-kestose administration on the intestinal flora of eels Eels were orally administered iKes fiber (1-kestose content 45.0% or more, manufactured by Itochu Sugar Co., Ltd.) to examine the effect on the intestinal flora of the eels as well as their total length and body weight. Tanks housing 200 eels each were used as control and test areas. Eel compound feed (mash, manufactured by Nippon Nosan Kogyo Co., Ltd.) was used as feed, and the feeding rate was 0.5-1.5% by mass of the total fish body weight housed. The feed was mixed with cod liver oil and water in a ratio of 1:0.1:1.3, kneaded, and formed into a soft mochi shape. The feed for the test group was prepared by suspending 10% by mass of iKes fiber and 2 x 10 ¹¹ cells/g of lactic acid bacteria (Lactobacillus plantarum FM8, Noriyoshi Co., Ltd.) in water at 0.3% by mass, and then mixing with compound feed and cod liver oil. Feeding was performed once a day at 9:00 on weekdays. After rearing for one month, all individuals were taken from each of the control and test groups, and their total length and weight were measured. From these, 20 individuals from each group (10 individuals with a size close to the average and 10 individuals with a size larger than the average) were dissected, and the intestinal contents were collected and stored at -80°C.
The frozen intestinal contents were thawed on ice, and each sample was suspended in 4 M guanidium thiocyanate, 100 mM Tris-HCl (pH 9.0), 40 mM EDTA, and pulverized with zirconia beads using a FastPrep FP100A instrument (MP Biomedicals, USA). DNA was extracted from the bead-treated suspension using a QIA QUBE. The V3-V4 region of the 16S rDNA sequence of bacteria and archaea was amplified using universal PCR primers (F: 3V4f_MIX: ACACTCTTTCCCTACACGACGCTCTTCCGATCT-NNNNN-CCTACGGGNGGCWGCAG, R: V3V4r_MIX: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-NNNNN-GACTACHVGGGTATCTAATCC) using ExTag HS (TaKaRa). The amplified PCR products were sequenced using MiSeq, and the results were analyzed with QIIME2 (version 2022.2) to calculate the relative occupancy of each bacterium at the genus level. For the measurement of short-chain fatty acids, 1 mL of ice-cold PBS was added to the collected intestinal content sample, which was thoroughly stirred with a vortex mixer and centrifuged to collect the supernatant as an analysis sample. The collected supernatant was pretreated with a long-chain/short-chain fatty acid analysis labeling reagent (product number XARFAR02) manufactured by YMC Corporation, and analyzed by HPLC. The analytical column used was a Waters ACQUITY UPLC BEH C18 1.7 μm x 150 mm, and the solvent was adjusted to a ratio of acetonitrile:methanol:water = 24:12:64, and the pH was adjusted to 4-5 with dilute hydrochloric acid. The relative occupancy rate and short-chain fatty acid concentration of each bacterium in the control and test groups were compared using the Mann-Whitney test, with p<0.1 being considered significant. The results are shown in Figure 1 (relative occupancy rate of bacteria) and Figure 2 (short-chain fatty acid concentration). Table 1 shows the feed efficiency calculated based on the weight gain in the control and test groups.

As shown in Figure 1, there was a significant increase in Romboutsia bacteria in the test group compared to the control group. Meanwhile, there was a significant decrease in Edwardsiella and Flavobacterium bacteria. Also, Figure 2 confirms that the concentrations of lactic acid and acetic acid in the intestine increased significantly in the test group. From these results, it was found that administering 1-kestose to eels increased the beneficial bacteria Romboutsia in the intestine of the eels, and increased short-chain fatty acids such as acetic acid and lactic acid. Meanwhile, there was a decrease in Edwardsiella bacteria, which are the causative bacteria of paracolo disease, and Flavobacterium bacteria, which are the causative bacteria of columnaris disease.

Example 2
Growth Effect of Various Carbohydrates on Bacteria of the Genus Romboutsia and Edwardsiella The assimilation of various carbohydrates by bacteria of the genus Romboutsia and Edwardsiella was examined.
Hypopolypeptone (Nissui Pharmaceuticals) 4.0 g, yeast extract (BD) 4.0 g, sodium chloride (Fujifilm Wako Pure Chemical Industries) 0.2 g, dipotassium hydrogen phosphate (Fujifilm Wako Pure Chemical Industries) 0.08 g, potassium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries) 0.08 g, magnesium sulfate heptahydrate (Fujifilm Wako Pure Chemical Industries) 0.02 g, calcium chloride dihydrate (Fujifilm Wako Pure Chemical Industries) 0.02 g, sodium bicarbonate (Wako Pure Chemical Industries) 4.0 g, bile powder (Fujifilm Wako Pure Chemical Industries) 1.0 g, Tween (Tokyo Chemical Industry Co., Ltd.) 4.0 g, vitamin K (Tokyo Chemical Industry Co., Ltd.) 20 uL, hemin (Fujifilm Wako Pure Chemical Industries) 0.01 g, resasurin sodium salt (Tokyo Chemical Industry Co., Ltd.) 1 mg, and cysteine hydrochloride 1.0 g (Tokyo Chemical Industry Co., Ltd.) were dissolved in 1 L of water to prepare a basal medium.
As the carbohydrate carbon source, sucrose (Suc, Fujifilm Wako Pure Chemical Industries), kestose (Kes, Itochu Sugar), galactooligosaccharide (Gos, Fujifilm Wako Pure Chemical Industries), inulin (Sigma-Aldrich), Fiberixa (FRX, Hayashibara), raffinose (Tokyo Chemical Industry Co., Ltd.), and CI-dextrin (CI-dex, Nisshin Sugar Co., Ltd.) were adjusted to a concentration of 50 mg/mL. A mixture of 500 μL of the basal medium and 500 μL of the adjusted carbohydrate solution was used as the culture medium. The bacteria used for the culture were Romboutsia lituseburenisis (JCM1404 strain) and Edwardsiella tarda (NBRC 105688 strain). These were pre-cultured in GAM medium (Nissui Pharmaceuticals), and 20 uL was inoculated into the culture solution. The turbidity at an absorbance of 660 nm of the medium before inoculation was taken as the turbidity at the start of the culture, and the turbidity was measured 24 hours after inoculation. The turbidity was measured using a plate reader (TECAN). The pH of the medium was also measured 24 hours after inoculation. The results are shown in Table 2.

As shown in Table 2, it was confirmed that bacteria of the genus Romboutsia grow by assimilating sucrose and 1-kestose and reduce the pH of the medium. On the other hand, it was confirmed that bacteria of the genus Edwardsiella grow in this medium composition when glucose is used as a carbon source (see Example 3), but growth was not confirmed with any of the oligosaccharides shown in Table 2, and no decrease in the pH of the medium was confirmed. From these results, it was revealed that bacteria of the genus Romboutsia efficiently assimilate sucrose and 1-kestose and reduce the pH of the medium, but Edwardsiella cannot utilize any of the carbohydrates.

Example 3
Comparison of growth of Romboutsia bacteria and Edwardsiella bacteria depending on medium pH The effect of the pH of the medium, which decreases by culturing Romboutsia bacteria, on the growth of Edwardsiella bacteria was evaluated. Glucose was used as a carbon source available to Edwardsiella bacteria. A medium was prepared by mixing 500 uL of the basal medium with the same composition as in Example 2 and a 50 mg/mL glucose solution. Acetic acid was added to the medium to adjust the final concentration to 0 mM, 5 mM, 10 mM, and 20 mM. Romboutsia lituseburensis (JCM1404 strain) and Edwardsiella tarda (NBRC 105688 strain) were pre-cultured in GAM medium (Nissui Pharmaceuticals), and 20 uL was inoculated into the culture solution. The turbidity at an absorbance of 660 nm of the medium before inoculation was taken as the turbidity at the start of the culture, and the turbidity was measured 24 hours after inoculation. The pH of the medium was also measured at the start of the culture (0 h) and 24 hours after inoculation (24 h).

As shown in Table 3, the growth of Romboutsia bacteria was slightly slowed at an acetic acid concentration of 20 mM, but growth was confirmed at all acetic acid concentrations. Compared with the pH at the beginning of the culture (immediately after inoculation), it was found that at a pH of about 7.5 to 6.3, acetic acid was produced as the growth of Romboutsia progressed, decreasing the pH of the medium to around 5.5.
In contrast, it was found that Edwardsiella bacteria grow without being affected by acetic acid at an acetic acid concentration of 0 to 10 mM, but their growth slows down at 20 mM, suggesting that the growth of Edwardsiella bacteria is suppressed when the pH at the initial stage of culture is 5.5 or lower.
From the above, it was presumed that the acetic acid produced in association with the proliferation of Romboutsia bacteria in the living body of fish lowers the pH of the surroundings, and as a result, the growth of Edwardsiella bacteria is suppressed. That is, from Examples 2 and 3, it was revealed that the efficient utilization of sucrose and 1-kestose by Romboutsia bacteria promotes the production of acetic acid in the intestines of fish, lowering the pH, and relatively suppressing the growth of Edwardsiella.

Example 4
Cultivation test of Edwardsiella bacteria using culture supernatant of Romboutsia bacteria The following test was carried out to verify whether organic acids such as acetic acid produced by culturing Romboutsia bacteria directly inhibit the growth of Edwardsiella bacteria.
First, 2 mL of glucose adjusted to 50 mg/mL and 2 mL of sterile water were added to 10 mL of the same basal medium as in Example 2 to prepare a medium. Then, 1800 μL was dispensed into three tubes, two of which were inoculated with live bacteria of Romboutsia lituseburensis (JCM1404 strain), and one was inoculated with dead bacteria of Romboutsia lituseburensis as a control. The dead bacteria were adjusted by heating the culture solution of Romboutsia lituseburensis in a heat block at 100 ° C for 15 minutes. The inoculation amount was 36 μL, and the culture was performed at 37 ° C under anaerobic conditions. The culture medium was collected immediately after inoculation of the live bacteria (0 days of culture) and one day after culture, and the culture medium was also collected immediately after inoculation of the dead bacteria. The culture medium was centrifuged at 15,000 rpm for 3 minutes to precipitate the bacterial cells, and the centrifuged supernatant was filtered through a 0.45 μm membrane filter to collect the culture supernatant, and the pH was measured.
The culture supernatants of these Romboutsia lituseburensis were used to culture Edwardsiella bacteria.
Specifically, 50 μL of distilled water was added to 950 μL of culture supernatant from day 0 of live and dead bacteria culture to make the total volume 1 mL, and 50 μL of 50 mg/mL glucose was added to 950 μL of culture supernatant from day 1 of live bacteria culture to make the total volume 1 mL, and 20 μL of Edwardsiella tarda (NBRC 105688 strain) was inoculated thereto and cultured at 37° C. under anaerobic conditions. The results are shown in FIG. 3.

The growth rate of Edwardsiella bacteria in the culture supernatant using killed Romboutsia bacteria as a control was set to 100%, and the growth rate of Edwardsiella for the other two groups was also expressed.
When the culture supernatant of the Romboutsia bacteria was used after 1 day of cultivation, the growth rate of the Edwardsiella bacteria was greatly reduced after 24 hours and 48 hours of cultivation. The growth of the Edwardsiella bacteria was suppressed due to the influence of the pH of the medium supernatant being greatly reduced by culturing the Romboutsia bacteria for 1 day. In other words, it is presumed that the culture product (short chain fatty acid) of the Romboutsia bacteria is involved in the growth inhibition of Edwardsiella.

Example 5
DNA was extracted from the intestinal contents of farmed fish (carp and tilapia) in the same manner as in Example 1. Meanwhile, a primer set (5'- TAAGCT TGACATCCTTTTTGACCTCTC-3', 5' - GCCTCACGACTTGGCTG-3') for detecting Romboutsia bacteria by PCR was designed. This primer set was designed to quantify Romboutsia lituseburensis (chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.jstage.jst.go.jp/article/bmfh/38/2/38_18-023/_pdf) by adding the underlined bases to the corresponding sequence of the 16S rRNA gene of Romboutsia lituseburensis at the 3' end of each primer set. In addition, a primer set (5'- GGAAGGTGTGCGTGTTAATAGCA -3', 5'- GAGACTCTAGCTTGCCAGTCTTGGA -3') was designed to detect Edwardsiella bacteria by PCR. This primer set was designed by aligning the top three 16S rRNA gene sequences derived from Edwardsiella bacteria frequently detected in the eel intestine.
Using these primer sets, PCR was performed on DNA extracted from the intestinal contents of each farmed fish, and on genomic DNA obtained by crushing the culture solution of Romboutsia lituseburensis (JCM1404 strain) and Edwardsiella tarda (NBRC 105688 strain) in the same manner as in Example 1. The PCR reaction solution was prepared according to the specifications of Quick Taq HS DyeMix (TOYOBO), and the reaction conditions were 40 cycles of 94 ° C. for 15 seconds, 60 ° C. for 15 seconds, and 72 ° C. for 20 seconds. Then, electrophoresis was performed using 2% agarose gel to confirm the amplified fragments.
As a result, in both cases where a primer set for detecting Romboutsia bacteria by PCR and a primer set for detecting Edwardsiella bacteria by PCR were used, amplified DNA fragments were observed in koi carp and tilapia, confirming the presence of Romboutsia bacteria or Edwardsiella bacteria.

Example 6
Effect of sucrose administration on eel feed efficiency Eels were orally administered sucrose (manufactured by Itochu Sugar Co., Ltd.) to examine the effect on feed efficiency, total length, and body weight. Aquariums containing 58 fish each were used as the control and test groups. Eel feed (mash, manufactured by Nippon Nosan Kogyo Co., Ltd.) was used as feed, and the feeding rate was 1.0-1.5% by mass of the total fish body weight. The feed was mixed with the feed, cod liver oil, and water in a ratio of 1:0.1:1.3, kneaded, and made into a soft mochi shape. The feed for the test group was prepared by dissolving sucrose in water at an additive rate of 10% by mass relative to the feed, and then mixing it with the feed and cod liver oil. Feeding was carried out once a day at 9:00 a.m. on weekdays. After 41 days of rearing, all individuals from each control and test group were taken and their total length and body weight were measured.
Table 4 shows the feed efficiency calculated based on the weight gain in the control and test groups.

As shown in Table 4, it was confirmed that administering sucrose improved feed efficiency.

The infectious disease preventive agent of the present invention uses a highly safe active ingredient, and can effectively prevent or treat bacterial infectious diseases by oral administration, making it extremely useful in aquaculture as a safe and simple infectious disease preventive and treatment agent that does not rely on antibiotics.

Claims (17)

An agent for preventing or treating infectious diseases in fish containing sucrose and/or 1-kestose as active ingredients. The agent for preventing or treating infectious diseases in fish according to claim 1, wherein the infectious disease is caused by infection with Edwardsiella bacteria and/or Flavobacterium bacteria. The preventive or therapeutic agent for infectious diseases in fish according to claim 1 or 2, wherein the fish is a freshwater fish. An intestinal regulator for fish containing sucrose and/or 1-kestose as active ingredients. The intestinal regulator for fish according to claim 4, which is based on the proliferation of bacteria of the genus Romboutsia in the intestines of fish. The intestinal regulator for fish according to claim 4, which is based on the effect of reducing Edwardsiella bacteria and/or Flavobacterium bacteria in the intestines of fish. The intestinal regulator for fish according to any one of claims 4 to 6, wherein the fish is a freshwater fish. An agent for promoting the proliferation of Romboutsia bacteria in the intestines of fish, containing sucrose and/or 1-kestose as active ingredients. An inhibitor of the proliferation of Edwardsiella and/or Flavobacterium bacteria in the intestines of fish, containing sucrose and/or 1-kestose as active ingredients. A feed efficiency improver containing sucrose and/or 1-kestose as active ingredients. A growth agent for Romboutsia bacteria containing sucrose and/or 1-kestose as active ingredients. A method for preventing or treating an infectious disease, comprising administering sucrose and/or 1-kestose to fish. The method for preventing or treating an infectious disease in fish according to claim 12, wherein the infectious disease is caused by infection with Edwardsiella bacteria and/or Flavobacterium bacteria. The method for preventing or treating an infectious disease in fish according to claim 12 or 13, wherein the fish is a freshwater fish. A method for improving the intestinal environment of fish, comprising administering sucrose and/or 1-kestose to the fish. The method for improving the intestinal environment of fish according to claim 15, which involves multiplying Romboutsia bacteria in the intestines of fish. The method for improving the intestinal environment of fish according to claim 15 or 16, which reduces Edwardsiella bacteria and/or Flavobacterium bacteria in the intestines of fish.

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