Animal disease prevention and control method suitable for underwater environment

Abstract

The invention discloses an animal disease prevention and control method suitable for an underwater environment, and belongs to the technical field of aquaculture. According to the invention, carbon dot (CDs)-mediated specific ASO delivery and adhesive hydrogel positioning capability are integrated, and the challenge of delivery of ASO to pathogenic bacteria in water wound care is solved. The CDs with the diameter of about 3 nanometers synthesized by the invention have excellent membrane penetrability and almost no influence on bacterial activity, so that ASO can be delivered into bacterial cells without additional chemical promoters or equipment. The ASO / CDs are combined with the underwater adhesive hydrogel, so that the ASO / CDs compound can be locally and continuously released at an infected part, the continuation of a treatment effect is ensured, and bacterial infection diseases of aquaculture animals, such as fin rot of turbots, are effectively prevented.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture, and in particular, relates to an animal disease prevention and control method suitable for underwater environments. Background Art

[0002] Bacterial infections in underwater environments pose a major challenge to the health of humans and aquatic organisms. In terms of human health, it is extremely difficult to treat bacterial infections in wounds that are immersed in water for a long time (such as when bathing or swimming), because continuous water contact can wash away conventional treatments and hinder the delivery of effective drugs. In aquatic ecosystems, similarly, bacteria can colonize open wounds in marine organisms, often leading to severe infections, impeded healing, and in some cases, mass mortality. Traditionally, antibiotics have been the main means of preventing and treating diseases. However, the application of antibiotics in underwater environments faces many limitations. Antibiotics often cannot be maintained for local use, leading to environmental pollution and promoting the emergence of antibiotic-resistant bacterial strains. For emerging antimicrobial nanomaterials, they also lack specificity and targeting capabilities in underwater treatments. These issues further exacerbate the impact of drug residues on a wide range of aquatic environments. Therefore, developing an innovative method that can overcome these limitations and achieve underwater targeted wound treatment remains a challenge for microbiologists and marine biotechnologists.

[0003] Gene silencing based on antisense oligonucleotides (ASOs) is a promising alternative approach that can be used to specifically regulate bacterial genes. However, applications in underwater environments remain challenged by the efficient delivery of nucleic acids to bacterial cells. Traditional delivery methods, such as electroporation and chemical transformation, strictly rely on specialized laboratory operations, have limited effectiveness in water, or require complex and impractical equipment, limiting the potential for local bacterial gene regulation in underwater environments. Although nanoparticles such as gold nanoparticles and silicon nanoparticles have proven effective in delivering various payloads into eukaryotic cells via endocytosis, the ability to deliver nucleic acids into bacteria is limited by the bacterial cell wall. In addition, the dynamic nature of aquatic ecosystems further increases the difficulty of delivery. The presence of water flow, temperature changes, and diverse microbial communities can all affect the local concentration and thus the efficacy of ASOs, making delivery a significant challenge.

[0004] Therefore, innovative delivery systems are urgently needed to enhance the uptake of ASOs into bacterial cells while maintaining their local concentrations in water. Summary of the invention

[0005] The purpose of the present invention is to provide an animal disease prevention and control method suitable for underwater environments to make up for the deficiencies of the prior art.

[0006] To achieve the above object, the present invention is achieved through the following technical solutions: A method for preventing and controlling animal diseases in an underwater environment, comprising the following steps: (1) Preparation of antisense oligonucleotide / carbon dot complexes (ASO / CDs); (2) verifying through experiments that the ASO / CDs can silence specific genes of target bacteria; (3) Preparation of underwater viscous hydrogel; (4) Treat the wounds of aquatic animals infected with bacteria with ASO / CDs. Each time the wound is cleaned with sterile water, it is immediately sealed with underwater adhesive hydrogel until the bacterial infection is cured.

[0007] Furthermore, the step (1) includes: ① Synthesis of carbon dots: citric acid and polyethyleneimine were dissolved in ultrapure water and ultrasonically treated; then, the mixed solution was transferred to a reactor water bath and reacted at 200°C. After the reaction was completed, it was cooled to room temperature and then the yellow solution was dialyzed in ultrapure water using a dialysis bag; finally, the filtrate was vacuum freeze-dried to obtain yellow carbon dot powder for later use; ②CDs loaded with ASO: CDs and ASO were incubated in ultrapure water at a certain mass ratio to obtain an ASO / CDs complex.

[0008] Furthermore, in step (2): ASO / CDs are added to a bacterial suspension and the mixture is incubated; after incubation, the incubated bacterial suspension is inoculated on an LB agar plate containing skimmed milk powder and NaCl, and then incubated again; and the silencing effect of ASO / CDs on specific genes of bacteria is evaluated by observing the transparent zone around the bacterial colonies.

[0009] Furthermore, in the step (3): tannic acid, chitosan and aluminum nitrate nonahydrate are dissolved in water and stirred evenly, then acrylic acid and N,N'-methylenebisacrylamide are added under stirring and stirred evenly; finally, ammonium persulfate solution is slowly added dropwise, and ultrasonic treatment is performed to remove bubbles; the resulting mixture is poured into a mold and sealed, and the temperature is gradually increased from 60°C to 80°C in a water bath, and finally maintained at 80°C for 30 minutes before cooling to room temperature.

[0010] Furthermore, in step (4), the wounds of aquatic animals infected with bacteria are treated with ASO / CDs every day, with the mass ratio of CDs to ASO being 20:1, for 7 consecutive days, once every 12 hours.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention integrates the specific ASO delivery mediated by carbon dots (CDs) with the localization ability of adhesive hydrogels, solving the challenge of delivering ASO to pathogenic bacteria in wound care in water. The CDs synthesized in the present invention with a diameter of about 3 nanometers have excellent membrane permeability and almost no effect on bacterial activity, enabling them to deliver ASO to bacterial cells without the need for additional chemical promoters or equipment. The combination of ASO / CDs and underwater adhesive hydrogels enables the ASO / CDs complex to achieve local and sustained release at the site of infection, thereby ensuring the continuation of the therapeutic effect and effectively preventing bacterial infections in aquatic animals, such as fin rot in turbot. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 ASO / CDs against Vibrio anguillarum empA A diagram of the gene silencing effect.

[0013] Figure 2 This is the tensile properties of the hydrogel.

[0014] Figure 3 This is a graph showing the compression performance of the hydrogel.

[0015] Figure 4 This is a graph showing the adhesion performance of the hydrogel.

[0016] Figure 5 This is a graph showing the adhesion performance of hydrogel in seawater.

[0017] Figure 6 This is the result diagram showing that the viscosity of the hydrogel is basically not affected by the water flow rate.

[0018] Figure 7 are photos of turbot in different groups; among them, a is turbot not infected with Vibrio anguillarum; b is turbot with wounds infected with Vibrio anguillarum in the control group (treated with water); c is turbot treated with ASO / CDs-hydrogel complex; d is turbot group treated with oxolinic acid; e is turbot treated with CDs; f is turbot treated with ASO; g is turbot treated with hydrogel; h is turbot treated with ASO / CDs.

[0019] Figure 8 This is a graph showing the cytotoxicity results of the ASO / CDs-hydrogel complex. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below in conjunction with embodiments and drawings.

[0021] Embodiment 1: A method for preventing Vibrio anguillarum infection in animals (such as turbot) in an underwater environment, comprising the following steps: (1) Preparation of antisense oligonucleotide / carbon dot complexes (ASO / CDs): ① Synthesis of carbon dots: 1.0 g citric acid and 2.0 g polyethyleneimine were dissolved in 10 mL ultrapure water and ultrasonically treated for 10 min. Then, the mixed solution was transferred to a polytetrafluoroethylene-lined reactor for hydrothermal reaction at 200 °C for 2 h. After the reaction was completed, it was cooled to room temperature, and then the yellow solution was dialyzed in ultrapure water for 24 h using a dialysis bag (1000 Da). Finally, the filtrate was vacuum freeze-dried to obtain yellow carbon dot powder for later use.

[0022] ②CDs loaded with ASO: CDs and ASO were incubated in ultrapure water at a mass ratio of 20:1 (ASO: 500 nM, CDs: 580 μg / mL) for 30 min to obtain an ASO / CDs complex.

[0023] (2) Effect of ASO / CD on Vibrio anguillarum empA Gene silencing ASO / CDs (100 μL) were added to the Vibrio anguillarum suspension (900 μL, 10 6 CFU / mL), the final concentrations of ASO and CDs were 500 nM and 58 μg / mL, respectively. The mixture was incubated at 28°C for 2 h. The other Vibrio anguillarum groups were mixed with water (100 μL), CDs (100 μL, 580 μg / mL), ASO (100 μL, 5 mM) and c-DNA / CDs (100 μL, c-DNA: 500 nM, CDs: 570 μg / mL), and incubated at 28°C for 2 h. After incubation, 20 μL of the bacterial suspension from each group was inoculated on LB agar plates containing skimmed milk powder (3%) and NaCl (2%), and then incubated at 28°C for 24 h. The effect of ASO / CDs on Vibrio anguillarum was evaluated by observing the transparent zone around the bacterial colonies. empA Gene silencing effect. Figure 1 ) Result analysis: Compared with the Vibrio anguillarum treated with water (Control), CDs, ASO and c-DNA / CDs, the transparent zone around the bacteria treated with ASO / CDs was significantly smaller, proving that ASO / CDs can enter Vibrio anguillarum and inhibit its empA Gene expression.

[0024] (3) Preparation of underwater viscous hydrogel: Tannic acid (0.3 g), chitosan (0.3 g) and aluminum nitrate nonahydrate (80.0 mg) were dissolved in 10 mL of water and stirred evenly. Subsequently, acrylic acid (3.0 g) and N,N'-methylenebisacrylamide (10.0 mg) were added under stirring and stirred for 20 min. Finally, ammonium persulfate solution (34.8 mg / mL, 2.3 mL) was slowly added dropwise and ultrasonic treatment was performed to remove bubbles. The resulting mixture was poured into a mold and sealed. The temperature was gradually increased from 60°C to 80°C in a water bath, and finally maintained at 80°C for 30 min before cooling to room temperature.

[0025] 1. Characterization of mechanical properties of the underwater viscous hydrogel: ① Tensile properties: The hydrogel with a specification of 80×15×1 (mm³) was subjected to a tensile test at a speed of 5 mm / min on an electric test bench. Figure 2 As shown, the prepared hydrogel has good tensile properties and the elongation can reach 157%.

[0026] ②Compression performance: The hydrogel with a specification of 15×15×6 (mm³) was subjected to a 50% compression strain test at a speed of 5mm / min on an electric test bench. Figure 3 As shown, the prepared hydrogel has good compression properties, and the maximum stress at 50% compression deformation is 2000 kPa.

[0027] 2. Viscosity characterization of the underwater viscous hydrogel: ① Adhesion test of hydrogel to different substrates in air: Use hydrogel to bond two substrates together with a bonding area of ​​15×15mm². Apply finger pressure of about 1kPa to the bonding site for 30 seconds in room temperature air, and then remove the external pressure. After 1 minute, use the test bench to perform a shear stress test on the bonded substrates, and measure the maximum tensile stress required to break the bond point at a rate of 100mm / min, such as Figure 4 As shown, the hydrogel has good adhesion strength to biological tissue (pigskin), up to 50 kPa.

[0028] ② Adhesion of hydrogel to biological tissue (pigskin) at different times in seawater: After the pigskin adhered with hydrogel was immersed in seawater for different times (0, 0.5, 1, 1.5 and 2 h), shear stress test was performed according to the above method. Figure 5 As shown, the longer the time in seawater, the better the adhesion of the hydrogel to the biological tissue (pig skin).

[0029] ③ Adhesion strength of hydrogel to pigskin under simulated seawater conditions: The speed of the magnetic stirrer was adjusted (0, 500, 1000 and 1500 rpm / min) to simulate different seawater flow dynamics. After the pigskin adhered to the hydrogel was placed in a simulated seawater environment, a shear stress test was performed, such as Figure 6 As shown, the adhesion of the hydrogel to biological tissue (pigskin) is basically not affected by the water flow rate.

[0030] Embodiment 2: This embodiment is a method for preventing and treating white fin disease of turbot using ASO / CDs-hydrogel: Uniform, healthy and vigorous juvenile turbot were selected and randomly divided into eight groups, with 10 fish in each group. After the turbot were anesthetized with eugenol (100 mg / L), an incision was made along the dorsal fin using a sterilized scalpel to ensure that the length and depth of the wound were consistent. The first group of fish was uninfected, while the other seven groups were placed in a culture medium containing Vibrio anguillarum (LD 50 = 2 × 10 6 CFU / mL) for 24 h to induce infection. After infection, the fish were transferred to seawater without Vibrio anguillarum for continued breeding. The wounds of one group of infected fish were treated with ASO / CDs (ASO: 500 nM, CDs: 58 μg / mL, 50 μL) every day for 7 consecutive days, every 12 h. Each treatment was immediately sealed with hydrogel after cleaning the wound with sterile water. The remaining five groups were treated with sterile water (50 μL), CDs (58 μg / mL, 50 μL), ASO (500 nM, 50 μL), ASO / CDs (ASO: 500 nM, CDs: 58 μg / mL, 50 μL, CD to ASO mass ratio of 20:1) and hydrogel, respectively. The last group of fish was treated by immersion, immersed in seawater containing oxolinic acid (3 mg / L) for 3 hours every day for 7 consecutive days.

[0031] The results are as follows Figure 7 As shown: turbot not infected with Vibrio anguillarum showed strong vitality, and no signs of fin rot were observed after five days ( Figure 7 a). In contrast, turbot in the control group (treated with water) whose wounds were infected with Vibrio anguillarum showed symptoms such as whitening of the back and fin rot, and some individuals even died ( Figure 7 b). The vitality of turbot treated with ASO / CDs-hydrogel composites decreased slightly, but no fin rot occurred ( Figure 7 c), and no mortality was observed, similar to the group treated with oxolinic acid ( Figure 7 d). It is noteworthy that the results of the groups treated with CDs, ASO, hydrogel, or ASO / CDs alone were similar to those of the water-treated group ( Figure 7 eh), which indicates that the ASO / CDs-hydrogel composite can effectively prevent and treat white fin disease of turbot.

[0032] Embodiment 3: This example studies the cytotoxicity of ASO / CDs-hydrogel complexes: CSGC cells and HepG2 cells were used to determine the cytotoxicity of the ASO / CDs-hydrogel complex. ASO / CDs (ASO: 500 nM; CD: 58 μg / mL) and hydrogel (100 μg / mL) were incubated with CSGC cells and HepG2 cells for different time periods (0, 2, 4, and 8 h), and the CCK-8 kit was used to measure the cell activity after incubation to evaluate the cytotoxicity of the ASO / CDs-hydrogel complex. Figure 8 ) The results are as follows Figure 8 As shown, the ASO / CDs-hydrogel complex has low cytotoxicity.

[0033] Finally, although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preventing and controlling animal diseases in an underwater environment, characterized in that: The following steps are involved: (1) Preparation of antisense oligonucleotide / carbon dot complexes ASO / CDs; (2) Experiments have demonstrated that the ASO / CDs are capable of silencing specific genes of target bacteria; (3) Preparation of underwater viscous hydrogel; (4) Treat the wounds of aquatic animals infected with bacteria with ASO / CDs. Each time the wound is cleaned with sterile water, it is immediately sealed with underwater adhesive hydrogel until the bacterial infection is cured.

2. The animal disease prevention and control method according to claim 1, characterized in that: The step (1) comprises: ① Synthesis of carbon dots: citric acid and polyethyleneimine were dissolved in ultrapure water and ultrasonically treated; then, the mixed solution was transferred to a reactor water bath and reacted at 200°C. After the reaction was completed, it was cooled to room temperature and then the yellow solution was dialyzed in ultrapure water using a dialysis bag; finally, the filtrate was vacuum freeze-dried to obtain yellow carbon dot powder for later use; ②CDs loaded with ASO: CDs and ASO were incubated in ultrapure water at a certain mass ratio to obtain an ASO / CDs complex.

3. The animal disease prevention and control method according to claim 1, characterized in that: In the step (2), ASO / CDs are added to a bacterial suspension and the mixture is incubated; after incubation, the incubated bacterial suspension is inoculated on an LB agar plate containing skimmed milk powder and NaCl, and then incubated again; and the silencing effect of ASO / CDs on specific genes of bacteria is evaluated by observing the transparent zone around the bacterial colonies.

4. The animal disease prevention and control method according to claim 1, characterized in that: In the step (3), tannic acid, chitosan and aluminum nitrate nonahydrate are dissolved in water and stirred evenly, and then acrylic acid and N,N'-methylenebisacrylamide are added under stirring and stirred evenly; finally, ammonium persulfate solution is slowly added dropwise, and ultrasonic treatment is performed to remove bubbles; the obtained mixture is poured into a mold and sealed, and the temperature is gradually increased from 60°C to 80°C in a water bath, and finally maintained at 80°C for 30 minutes and then cooled to room temperature.

5. The animal disease prevention and control method according to claim 1, characterized in that: In the step (4), the wounds of aquatic animals infected with bacteria are treated with ASO / CDs every day, with the mass ratio of CDs to ASO being 20:1, for 7 consecutive days, once every 12 hours.

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