Use of allicin e for prevention and treatment of nocardia seriolae infection in largemouth bass

Abstract

A use of allicin E for prevention and treatment of Nocardia seriolae infection, comprising: adding a compound as represented by formula (I) to a feed for largemouth bass. The feed can ameliorate the condition of largemouth bass infected with Nocardia, reduce the mortality rate of largemouth bass, and reduce the threat to food and environmental safety caused by long-term use of antibiotics.

Description

Use of garlic E in preventing and treating Nocardia amberjack infection in largemouth bass. Technical Field

[0001] This invention relates to the field of Nocardia amberjack infection prevention and control technology, and proposes the use of garlic E to prevent and control Nocardia amberjack infection in largemouth bass. Background Technology

[0002] Nocardia is taxonomically classified as belonging to the domain Bacteria, phylum Firmicutes, class Actinomycetales, family Nocardiaceae, and genus Nocardia. It is an acid-resistant or partially acid-resistant, facultative anaerobic, Gram-positive bacterium, a zoonotic pathogen characterized by tissue suppuration, necrosis, or abscess formation. It can infect humans, horses, cattle, pigs, cats, dogs, rodents, and fish.

[0003] Nocardiosis in fish is a chronic, systemic, granulomatous disease, commonly known as "nodular disease." The main clinical symptoms of infected fish are bleeding and ulceration on the body surface and numerous white nodules covering the internal organs. To date, three pathogens of nocardiosis in fish have been isolated: Nocardia seriolae, Nocardia asteroides, and Nocardia salmonicida.

[0004] Related studies have shown that *Nocardia seriolae* has become a major pathogen causing nocardiosis in fish, with its prevalence increasing year by year in my country and Southeast Asia, and the number of infected fish species constantly expanding. According to incomplete statistics, *Nocardia seriolae* can infect more than 39 species of freshwater and marine fish, causing increasingly serious economic losses to the global aquaculture industry. Therefore, there is an urgent need in this field to develop novel antimicrobial substances targeting *Nocardia seriolae*.

[0005] Summary of the Invention

[0006] The purpose of this invention is to provide a novel antibacterial substance targeting Nocardia amberjack.

[0007] A first aspect of the present invention provides a use of garlic E, characterized in that it is used to prepare a feed composition for reducing or improving Nocardia infection in yellowtail amberjack; wherein the garlic E has a structure as shown in the following formula:

[0008] In another preferred embodiment, the feed composition is used to treat or improve diseases or symptoms caused by Nocardia infection in yellowtail.

[0009] In another preferred embodiment, the amount of garlic E in the feed composition is 100-2000 mg / kg of feed.

[0010] In another preferred embodiment, the allicin E in the feed composition is characterized by being used at an amount of 900-1100 mg / kg of feed.

[0011] In another preferred embodiment, the feed composition is used to increase the permeability of Nocardia amberjack cell membranes.

[0012] In another preferred embodiment, the feed composition is used to dissolve the cell membrane of Nocardia amberjack.

[0013] In another preferred embodiment, the feed composition is used to inhibit the formation of Nocardia amberjack biofilm.

[0014] In another preferred embodiment, the feed composition is used to reduce mortality in largemouth bass infected with Nocardia spp.

[0015] In another preferred embodiment, the garlic E ingredient is prepared as a stock solution with a concentration of 40-80 mg / L for use.

[0016] In another preferred embodiment, the garlic E ingredient is prepared as a stock solution with a concentration of 120-130 mg / L.

[0017] In a second aspect, the present invention provides a largemouth bass feed composition, characterized in that the feed composition comprises garlic E.

[0018] In another preferred embodiment, the content of allicin E in the feed composition is 100-2000 mg / kg feed.

[0019] In another preferred embodiment, the amount of garlic E in the feed composition is 900-1100 mg / kg of feed.

[0020] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0021] Figure 1 shows the inhibition zone of allicin E on Nocardia amberjack.

[0022] Figure 2 shows the MIC determination results of allicin E against Nocardia amberjack.

[0023] Figure 3 shows the results of the MBC assay for allicin E on Nocardia amberjack.

[0024] Figure 4 shows the inhibition rate of different concentrations of garlic E on Nocardia amberjack.

[0025] Figure 5 shows an electron microscope image of the cell structure of Nocardia amberjack after treatment with garlic E.

[0026] Figure 6 shows the effect of garlic E on the activities of lactate dehydrogenase and β-D-galactosidase in Nocardia amberjack.

[0027] Figure 7 shows the in vivo efficacy of allicin against Nocardia amberjack in largemouth bass. Detailed Implementation

[0028] Through long-term and in-depth research, the inventors discovered that adding garlic E to feed can effectively improve the infection status of Nocardia amberjack in largemouth bass, exhibiting good in vitro antibacterial activity against Nocardia amberjack, disrupting the cell structure of Nocardia amberjack, and increasing cell membrane permeability. Based on the above findings, the inventors completed this invention.

[0029] Garlic E

[0030] ALE (Allicin E) is a single compound synthesized biomimetically by modifying the structure outside the antibacterial active group of ordinary allicin. Its structure is shown in the following formula:

[0031] Garlic E has broad-spectrum bactericidal and green safety characteristics, and can effectively replace traditional antibiotics, supporting environmental disinfection, animal and plant protection, preservation, and upgrading of the biopharmaceutical industry.

[0032] Feed composition with added garlic E

[0033] This invention provides a feed composition; the feed composition is fortified with garlic E, the garlic E having the structure shown in the following formula:

[0034] Because garlic E has good bactericidal properties, the feed composition described above is used to reduce or improve the infection of largemouth bass with Nocardia amberjack. In a preferred embodiment, the amount of garlic E in the feed composition is 100-2000 mg / kg feed, more preferably 900-1100 mg / kg feed.

[0035] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0036] Example 1: Strain Culture

[0037] The experiment was conducted by the Zhejiang Provincial Freshwater Fisheries Research Institute. Nocardiella LY21811 was isolated from the kidneys of largemouth bass infected with nocardiosis and stored at -80℃. The strain LY21811 was removed from the refrigerator and streaked onto Brian Heart Infusion Agar (BHIA) to activate and revive the bacteria. Single colonies were picked and inoculated into test tubes containing 5 mL of BHI broth, and cultured with shaking at 28℃ and 150 rpm for 5–7 days. The bacterial concentration was determined by turbidimetric assay, and in subsequent experiments, the culture was diluted to the desired concentration using sterile BHI broth.

[0038] Example 2: Determination of inhibition zones

[0039] The inhibition zone was determined using the Oxford cup method. 100 μL of 1x10⁻¹⁰ solution was used. 8 CFU / mL Nocardia amberjack suspension was spread onto BHIA plates. 100 μL each of 1000 mg / L and 100 mg / L garlic E solution were added to the pre-drilled wells of the plates. The plates were incubated at 28℃ for 5 days. Once the plates were fully colonized, the diameter of the inhibition zone was measured.

[0040] Inhibition zone: The Oxford cup antibacterial experiment showed that the diameter of the inhibition zone of 1000 mg / L garlic E against Nocardia ferruginis was about 29 mm, indicating that garlic E has good in vitro antibacterial activity against this bacterium (Figure 1).

[0041] Example 3: In vitro antibacterial activity assay

[0042] The MIC of allicin E against Nocardia amberis was determined using the resazurin method. 100 μL of 1x10⁻⁶ g of allicin was added to each well of a 96-well plate. 5 CFU / mL Nocardia amberjack bacterial suspension was added to the first row of wells, followed by serial dilutions of 2-fold to achieve final drug concentrations of 1000, 5000, 250, 125, 62.5, 31.3, 15.6, and 7.8 mg / L. A positive control (100 μL bacterial suspension + 100 μL BHI broth) and a negative control (200 μL BHI broth) were also included, with 6 wells per group (6 replicates). The 96-well plate was incubated at 28°C for 5 days. 50 μL of resveratrol solution was added to each well, and the plate was incubated for another 24 hours. The color of the bacterial suspension was then observed (pink indicates bacterial growth, blue indicates inhibition). The lowest drug concentration at which no bacterial growth was observed was the MIC.

[0043] Based on the MIC experiment, 100 μL of bacterial suspension was taken from the clear well and spread onto BHI agar plates. The plates were incubated at 28°C for 24 h. The lowest concentration at which no colonies grew on the plate was taken as the MBC of the drug for that strain.

[0044] The inhibition rate of allicin E against Nocardia amberjack was determined using the CCK-8 assay. 200 μL of 1x10⁻⁶ allicin E was added to each well of a 96-well plate. 5 CFU / mL Nocardia amberjack bacterial suspension was used, followed by the addition of allicin at concentrations of 1 / 2 MIC, MIC, and 2 MIC. A negative control group (no drug, but bacterial suspension) and a blank control group (no drug, no bacterial suspension) were also established. The 96-well plates were incubated at 28°C for 5 days. 20 μL of CCK-8 reagent was added to each well, and the inhibition rate of each drug concentration against Nocardia amberjack was calculated. Cell inhibition rate = [(Ac-As) / (Ac-Ab)] x 100%, where AS represents the absorbance of the experimental groups (i.e., each drug group), Ac represents the absorbance of the positive control group, and Ab represents the absorbance of the blank control group.

[0045] The MIC: Resazurin test results showed that when the concentration of garlic E was greater than 62.5 mg, the bacterial culture turned blue (no cell activity) (Figure 2), indicating that the MIC value of garlic E for this bacterium was 62.5 mg / L.

[0046] MBC: Bacterial solutions with concentrations greater than 62.5 mg / L were aspirated from each well of a 96-well plate and spread. The results showed that no colonies grew when the concentration of allicin E was greater than 125 mg / L, indicating that the MBC concentration was 125 mg / L (Figure 3).

[0047] Inhibition rate: As shown in Figure 4, the CCK-8 assay results show that the activity of Nocardia amberjack is negatively correlated with the concentration of garlic E. The inhibition rate is 58.41% when the concentration is 1 / 2 MIC, and as high as 98.45% when the concentration is MIC.

[0048] Example 4 Electron Microscopic Observation

[0049] In containing 1x10 7 In test tubes containing CFU / mL Nocardia amberjack bacterial suspension, allicin E (i.e., drug treatment group) was added to a final concentration of 1 / 2 MIC. A control group with an equal amount of PBS was also set up. The suspensions were cultured at 28℃ and 180 rpm for 3 days. 1.5 mL of bacterial suspension was taken from each tube and centrifuged at 8000 rpm for 5 min at 4℃. The supernatant was discarded, and the precipitate was washed three times with sterile PBS. The precipitate was collected, and 2.5% glutaraldehyde was added. The tubes were then fixed at 4℃ for 12 h and sent to Hangzhou Yanqu Information Technology Co., Ltd. The morphology and structure of the bacteria were observed by transmission electron microscopy (TEM).

[0050] In the control group, the Nocardia amberjack cells maintained their intact structure, while in the garlic E-treated group, some bacterial cell membranes dissolved, resulting in leakage of large amounts of cell contents and vacuolization. (See Figure 5).

[0051] Example 5: Cell Membrane Permeability Measurement

[0052] In containing 1x10 7 CFU / mL Nocardia amberjack bacterial culture was added to test tubes with allicin E at final concentrations of 1 / 2 MIC, MIC, and 2 MIC (the drug treatment groups). A control group with an equal volume of PBS was also included. The cultures were incubated at 28℃ and 180 rpm in a shaker. Bacterial culture was collected at days 0, 2, 4, 6, 8, 10, 12, and 14. The supernatant and bacterial pellet were obtained by centrifugation. The supernatant was used to determine protein extravasation (Coomassie Brilliant Blue method), and the bacterial pellet was used to determine lactate dehydrogenase activity (DH kit, Nanjing Jiancheng Bioengineering Institute). On day 7, the bacterial pellet was used to determine BD-galactosidase activity (ONPG reagent, Beijing Bio-Lab Technology Co., Ltd.)

[0053] Nocardia amberjack was treated with three different concentrations (1 / 2MIC, MIC, and 2MIC) of garlic E. The results showed that the lactate dehydrogenase activity in all three garlic E treatment groups was lower than that in the control group within 14 days (Figure 6), indicating that garlic E inhibited the growth of Nocardia amberjack. The BD-galactosidase activity in each group was measured after 7 days of drug treatment. The results showed that the enzyme activity in all three drug treatment groups was significantly lower than that in the control group (Figure 6). Garlic E treatment increased cell membrane permeability, leading to leakage of β-D-galactosidase.

[0054] Example 6: In vivo antibacterial efficacy experiment

[0055] Largemouth bass were sourced from the experimental base of the Zhejiang Provincial Freshwater Fisheries Research Institute. The bass were uniform in size, weighing approximately 10g, and exhibited normal activity and feeding. Before the experiment, all fish were temporarily held for one week, during which the mortality rate was less than 0.5%. The water temperature was maintained at 25±1℃, and the fish were fed twice daily. Approximately 30% of the water was changed according to water quality indicators. Ten fish were randomly selected before the experiment for pathogen testing to ensure they did not carry common pathogens. Before the formal experiment, medicated feed was prepared by first crushing the feed, adding the medication, mixing thoroughly, pressing it into pellets using a pelleting machine, and then drying it before use.

[0056] Healthy largemouth bass were randomly divided into four groups: a test drug group (garlic E, 200, 400, 800, and 1000 mg / kg feed), a control drug group (enrofloxacin, 1000 mg / kg feed), a challenge control group, and a blank control group. Each group had three replicates, with 20 fish per replicate. Fish were fed for 2–3 days until all experimental fish were feeding and the feeding time was less than 1 minute before the drug administration experiment began. Considering that bass would eat less after challenge, this study used a pre-challenge approach. During the experiment, fish were fed twice daily (9:00 and 16:00). The test drug group and the control drug group were fed medicated feed, while the challenge control group and the blank control group were fed regular feed. Water was changed according to water quality indicators, with approximately 30% of the water replaced. Water quality parameters such as temperature, dissolved oxygen, pH, ammonia nitrogen, and nitrite nitrogen were measured daily. During the experiment, the water temperature was maintained at 25±1℃, dissolved oxygen greater than 5.0 mg, pH between 7.8 and 9.0, ammonia nitrogen less than 0.5 mg, and nitrite nitrogen less than 0.1 mg. Three days after drug administration, fish were challenged with the novel coronavirus. Based on preliminary experiments, the concentration of the challenge bacterial solution was determined to be 5 x 10 CFU / mL (mortality rate approximately 80%–100%). Each fish in the test drug group, control drug group, and challenge control group was intraperitoneally injected with 0.1 mL of *Nocardia amberjack* PBS resuspension. The blank control group was injected with an equal volume of PBS. Fish were observed continuously for 30 days after challenge, and mortality was recorded. The relative protection rate (RPS) was calculated using the following formula: RPS = (1 - mortality rate of the test group / mortality rate of the challenge control group) x 100%.

[0057] Largemouth bass were fed with feed containing different concentrations of garlic E before being challenged with Nocardia amberriformis. The results showed that within 30 days of challenge, the relative protection rates of 1000, 800, 400, and 200 mg / kg garlic E were 59.5%, 42.3%, 23.6%, and 14.7%, respectively, while the relative protection rate of the enrofloxacin-positive control group was 84.6% (Figure 7). These results indicate that adding garlic E can improve the resistance of largemouth bass to Nocardia amberriformis infection.

[0058] discuss

[0059] (1) Garlic E has good in vitro antibacterial activity against Nocardia amberjack, with MIC and MBC values ​​of 62.5 mg / L and 125 mg / L, respectively.

[0060] (2) Treatment of Nocardia amberjack with garlic E can destroy its cell structure, increase cell membrane permeability and cause leakage of substances such as BD-galactosidase.

[0061] (3) Feed with added garlic E can effectively reduce the mortality rate of Nocardia amberjack infection in largemouth bass.

[0062] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A use of garlic E, characterized in that, Feed compositions for preparing a reduction or amelioration of an infection of Nocardia seriolae in large-mouth bass; wherein the allicin E has the following structure:

2. The use as described in claim 1, characterized in that, The feed composition is used to treat or improve diseases or symptoms caused by Nocardia infection in yellowtail.

3. The use as described in claim 1, characterized in that, In the feed composition, the amount of garlic E is 100-2000 mg / kg of feed.

4. The use as described in claim 1, characterized in that, In the feed composition, the amount of garlic E is 900-1100 mg / kg of feed.

5. The use as described in claim 1, characterized in that, The feed composition described herein is used to increase the permeability of Nocardia amberjack cell membranes. In another preferred embodiment, the feed composition is used to dissolve the cell membrane of Nocardia amberjack.

6. The use as described in claim 1, characterized in that, The feed composition is used to inhibit the formation of Nocardia amberjack biofilm.

7. The use as described in claim 1, characterized in that, The feed composition described herein is used to reduce mortality in largemouth bass infected with Nocardia spp.

8. A largemouth bass feed composition, characterized in that, The feed composition includes garlic E.

9. The composition according to claim 8, characterized in that, The content of allicin E in the feed composition is 100-2000 mg / kg feed.

10. The composition according to claim 8, characterized in that, In the feed composition, the amount of garlic E is 900-1100 mg / kg of feed.

Images