Application of aeromonas hydrophila Ah-221212 strain in preparation of vaccine for preventing septicemia of aquatic animals caused by aeromonas hydrophila

Abstract

The invention provides an application of an aeromonas hydrophila Ah-221212 strain in preparation of a vaccine for preventing septicemia of aquatic animals caused by aeromonas hydrophila. The inactivated vaccine prepared from the specific aeromonas hydrophila Ah-221212 strain has the advantages of simple preparation steps and low cost, can have multiple functions of targeted delivery, self-adjuvant effect, slow release and the like, does not need to additionally add traditional adjuvants such as an aluminum adjuvant and an oil adjuvant, and is a novel aeromonas hydrophila aeromonas hydrophila vaccine with excellent performance.

Description

Technical Field

[0001] This invention belongs to the field of vaccine preparation technology, specifically relating to the application of Aeromonas hydrophila strain Ah-221212 in the preparation of a vaccine to prevent septicemia in aquatic animals caused by Aeromonas hydrophila. Background Technology

[0002] Currently, China has a large production of freshwater farmed fish. However, freshwater fish farming is plagued by bacterial septicemia, primarily caused by *Aeromonas hydrophila*. *Aeromonas hydrophila* is a common pathogen in aquaculture, an important Gram-negative opportunistic pathogen. Its pathogenicity is attributed to a combination of virulence factors, including hemolysins, cell-excitatory enterotoxins, and extracellular enzymes. This opportunistic pathogen can cause bacterial septicemia, enteritis, and ascites in various aquatic animals, resulting in significant economic losses for the aquaculture industry. Chronic infection manifests as ulcerative lesions characterized by redness, swelling, inflammation, and hemorrhage. Acute infection may lead to symptoms before death such as bulging and ruptured eyeballs, skin reddening, abdominal swelling with ascites, and rotting of the gill filaments. Current prevention methods primarily include the use of antibiotics and environmental modifiers, but these methods can lead to increased drug resistance and aquatic pollution.

[0003] Vaccination is an effective and environmentally friendly means of disease control. However, currently used inactivated Aeromonas hydrophila septicemia vaccines have limitations such as low antigen delivery efficiency and adjuvant-dependent immunogenicity. Therefore, it is essential to design a novel vaccine that combines targeted delivery, self-adjuvant effect, and sustained-release function.

[0004] The Yangtze sturgeon, a medium-sized economic fish species in the upper and middle reaches of the Yangtze River, is currently classified as "Extinct in the Wild" by the International Union for Conservation of Nature (IUCN), indicating a very small population. Almost all Yangtze sturgeon individuals currently live in captivity (breeding and release stations, research institutions, etc.). However, in high-density captive breeding environments, septicemia caused by Aeromonas hydrophila is highly likely to break out, leading to large-scale mortality. Effective vaccines can significantly reduce mortality rates, ensuring the survival rate of these valuable captive-bred broodstock and juveniles, and providing a sufficient source of healthy seedlings for breeding and release. Furthermore, releasing unvaccinated, vulnerable juveniles directly into complex natural environments teeming with pathogens results in a very low survival rate.

[0005] Therefore, developing a vaccine against Aeromonas hydrophila in Yangtze sturgeon is of great significance for the prevention and / or treatment of septicemia caused by Aeromonas hydrophila, and will help promote the further development of aquaculture and ecological balance in Yangtze sturgeon and other aquaculture industries. Summary of the Invention

[0006] To address the problems and shortcomings of existing technologies, this invention provides the application of Aeromonas hydrophila strain Ah-221212 in the preparation of a vaccine to prevent septicemia in aquatic animals caused by Aeromonas hydrophila. The inactivated vaccine prepared from this specific Aeromonas hydrophila strain Ah-221212 has a simple preparation process, low cost, and can combine multiple functions such as targeted delivery, self-adjuvant effect, and sustained release. Furthermore, it does not require the addition of traditional adjuvants such as aluminum adjuvants or oil adjuvants, making it a novel and high-performance Aeromonas hydrophila vaccine.

[0007] This invention provides the application of Aeromonas hydrophila strain Ah-221212 in the preparation of a vaccine to prevent septicemia in aquatic animals caused by Aeromonas hydrophila. The accession number of Aeromonas hydrophila strain Ah-221212 is GDMCC No:66953, the accession address is Guangdong Provincial Center for Microbial Culture Collection, and the accession date is September 11, 2025.

[0008] The Aeromonas hydrophila vaccine provided by this invention is an inactivated vaccine, using Aeromonas hydrophila strain Ah-221212 as the active ingredient. It can effectively reduce local infections (such as skin ulcers, fin and tail rot, furuncles, and gill infections), enteritis (such as anal swelling and protrusion; ascites in the abdominal cavity; intestines empty of food, only yellow mucus, and intestinal wall congestion and inflammation) and septicemia (such as systemic hemorrhage, necrosis of internal organs, and ascites) caused by Aeromonas hydrophila. Modern aquaculture, especially with its high-density artificial breeding, is more susceptible to Aeromonas hydrophila infection and the aforementioned symptoms, particularly septicemia. The Aeromonas hydrophila vaccine provided by this invention can effectively treat and prevent the aforementioned local infections, enteritis, and septicemia caused by Aeromonas hydrophila, especially effectively reducing the incidence of septicemia and the mortality rate of infected animals.

[0009] Furthermore, relevant experiments have verified that this vaccine can effectively improve the survival rate of Yangtze sturgeon after being challenged with Aeromonas hydrophila. Moreover, the vaccine has no obvious toxicity to Yangtze sturgeon, and no significant pathological changes were observed within 28 days of administration, demonstrating a high level of safety.

[0010] It should be noted that selecting a bacterial strain for inactivated vaccine preparation is a complex process that requires comprehensive consideration of multiple factors, including pathogen characteristics, immune mechanisms, and safety. Therefore, selecting a suitable Aeromonas hydrophila strain as the active ingredient in vaccine preparation requires extensive experimental verification, and this selection process is extremely challenging. For example, firstly, there is the issue of immunogenicity. Some bacteria have poor immunogenicity in their surface structures; after inactivation and injection into the body, the immune system responds weakly and cannot generate sufficient protection. However, the Aeromonas hydrophila strain Ah-221212 selected in this invention possesses highly immunogenic surface antigens. Secondly, there is the issue of protective immune mechanisms. The immune response elicited by the vaccine must match the body's natural immune mechanism against the pathogen. The immune response elicited after inactivation of the Aeromonas hydrophila strain Ah-221212 selected in this invention matches the body's natural immune mechanism against the pathogen effectively. Third, regarding safety, Aeromonas hydrophila may produce toxins such as aerolysins, proteases, and endotoxins. Effective inactivation is crucial for vaccine use. The Aeromonas hydrophila strain Ah-221212 selected in this invention can be effectively inactivated under certain inactivation conditions, thus exhibiting reliable safety performance. Furthermore, under specific inactivation conditions, the Aeromonas hydrophila strain Ah-221212 selected in this invention can maintain its key protective antigen structure, ensuring the effectiveness of the subsequent vaccine.

[0011] Preferably, the symptoms of sepsis include at least one of acute death, organ necrosis, and hemorrhage.

[0012] Preferably, the above-mentioned vaccine (Aeromonas hydrophila vaccine) is used on aquatic animals, including sturgeon.

[0013] Preferably, the aquatic animal is the Yangtze sturgeon.

[0014] Preferably, the preparation method of the above-mentioned vaccine is as follows: S1. Preparing a primary strain using Aeromonas hydrophila strain Ah-221212; S2. Preparing a secondary strain using the primary strain; S3. Further culturing the secondary strain and then resuspending it at 10 °C. 8 ~10 10 S3. The bacterial solution was inactivated and ultrasonically disrupted. S4. The disrupted bacterial solution obtained in S3 was emulsified to obtain an emulsified disrupted bacterial solution. S5. A chitosan solution was prepared and mixed with the emulsified disrupted bacterial solution to coat the disrupted bacterial cells and obtain the vaccine.

[0015] Preferably, the specific operation of S1 is as follows: Take the frozen strain of Aeromonas hydrophila Ah-221212 and inoculate it into the brain heart extract culture medium, and incubate it at 26-32℃ for 12-36 hours. Pick typical single colonies, inoculate them into the brain heart extract culture medium, and incubate them at 26-32℃ for 6-24 hours. After the purity is tested and found to be qualified, it is used as the primary strain.

[0016] Preferably, the primary strain is stored at 2-8℃ for no more than 7 days. Preferably, the primary seed strain is passaged no more than 5 times on the culture medium.

[0017] Preferably, the specific operation of S2 is as follows: take the primary strain and inoculate it into the brain and heart infusion culture medium, and culture it at 26-32℃ and 200-400 rpm for more than 48 hours. After testing its purity, it is used as the secondary strain.

[0018] Preferably, the secondary strains are stored at 2-8℃ for no more than 7 days.

[0019] Preferably, the specific operation of S3 is as follows: S31. Take secondary bacterial strain and inoculate brain and heart extract culture medium at a volume ratio of 6%-10%. After incubation at 26-32℃ and shaking at 200-400 rpm for no less than 48 hours, centrifuge at 4500-5500 rpm for 10-20 minutes, resuspend in PBS, count with a hemocytometer, and dilute to 10⁻¹⁰. 8 ~10 10 S32. Add 0.45-0.55% formaldehyde to the bacterial culture prepared in S31 to inactivate it for 44-52 hours. Centrifuge the successfully inactivated bacterial culture at 4500-5500 rpm for 10-20 minutes and resuspend it. Wash it with PBS 2-4 times. Then, sonicate the bacterial culture at 100-400 watts for 10-60 minutes.

[0020] Preferably, in step S32, if no colony growth is observed after the formaldehyde-inactivated bacterial solution is applied to the brain and heart infusion culture medium for 44-52 hours, it indicates successful inactivation.

[0021] Preferably, the broken bacterial solution obtained after the crushing is completed is stored at 4±0.5℃.

[0022] Preferably, the specific operation of S4 is as follows: double-distilled water, medium-chain triglycerides, Tween 20, and broken bacterial solution are mixed and emulsified in a volume ratio of 50~70:22~32:6~10:4~6.

[0023] Preferably, the emulsification time is 0.5 to 2 hours.

[0024] Preferably, in S5, the average particle size of the Aeromonas hydrophila vaccine particles is 230.57±13.8 nm.

[0025] Preferably, the vaccine particles are chitosan nanoparticles carrying broken cells of Aeromonas hydrophila strain Ah-221212.

[0026] Preferably, the specific operation of S5 is as follows: S51. Dissolve chitosan in an acetic acid solution with a volume fraction of 0.5~1.5% to prepare a chitosan solution with a concentration of 8~12mg / mL; S52. Add emulsified and broken bacterial solution to the chitosan solution at a volume ratio of 1~1.5:1~1.5, and stir at a constant temperature for 1~6 hours to fully coat the broken bacterial cells.

[0027] Preferably, in step S51, during the preparation of the chitosan solution, the chitosan is dissolved in an acetic acid solution with a volume fraction of 0.5-1.5%, and then stirred at a constant temperature for 4-12 hours until the solution becomes clear.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This vaccine has low production costs and relatively low technical barriers. Chitosan nanocarriers physically encapsulate antigens, protecting them from degradation by the fish's mucus layer, digestive enzymes, or environmental factors (such as pH and temperature), significantly improving antigen delivery efficiency in target tissues (such as the intestines, gills, or immune cells). Through the sustained-release design of the nanocarriers, antigens can be continuously released within the fish, prolonging the exposure time of the immune system, inducing long-lasting immune memory, and enhancing the long-term effect of the vaccine.

[0029] 2. This vaccine can prevent bacterial septicemia in sturgeon caused by Aeromonas hydrophila. According to relevant experiments, the relative immune protection rate is greater than 75%, and the safety is good. It will not stimulate the body to produce pathological changes. It is particularly suitable for the prevention of bacterial septicemia in sturgeon such as Yangtze sturgeon in actual production, reducing economic losses in the aquaculture production process. Therefore, it is conducive to increasing the total number of Yangtze sturgeon in the ecosystem and promoting the balance and stability of the ecosystem. Attached Figure Description

[0030] Figure 1 The colony morphology of Aeromonas hydrophila strain Ah-221212.

[0031] Figure 2 This is a microscopic examination of the *Aeromonas hydrophila* strain Ah-221212 after Gram staining.

[0032] Figure 3 This is a particle size distribution diagram of the chitosan solution, broken bacterial solution, and vaccine solution in Example 2.

[0033] Figure 4 The images are electron microscope images of the bacterial solution (a) before inactivation and disruption in S32, the disrupted bacterial solution (b) after emulsification in S4, and the vaccine solution (c) in S52 in Example 2. Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0035] Example 1: Isolation and identification of Aeromonas hydrophila strain Ah-221212 1. Strain isolation: The pathogenic material was obtained from a farmed Chinese sturgeon from the Shanghai Aquatic Wildlife Conservation Center on December 12, 2022. The sturgeon weighed 722.2g and measured 48cm in length. Liver, spleen, kidney, gills, and intestinal tissues were collected from near-death sturgeon exhibiting typical symptoms. Ascites fluid, valvular intestine, and gill tissue were collected using a sterile inoculation loop and streaked onto brain and heart infusion medium. After incubation at 30°C for 24 hours, single colonies were picked for secondary isolation and purification. The purified colonies were grayish-white, smooth, and slightly convex. Figure 1 As shown. Microscopic examination after Gram staining revealed that the bacterium stained red, indicating it is a Gram-negative bacterium. Under the microscope, it appears as a short rod-shaped bacterium, as shown. Figure 2 As shown, the bacteria have blunt, rounded ends, pili, and a single flagellum, indicating they are motile. Single colonies were then inoculated into fish brain heart extract culture medium for further subculturing.

[0036] 2. Strain identification: Identification was performed using both physiological and biochemical methods and 16S rRNA sequencing.

[0037] Fresh bacterial culture was added according to the instructions of the API 20E biochemical kit. The isolated strain was identified as Aeromonas hydrophila using the ATB automated microbial identification instrument and in conjunction with Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria.

[0038] DNA was extracted from the purified bacterial strain. Universal primers for the bacterial 16S rRNA gene were synthesized by Sangon Biotech (Shanghai) Co., Ltd.: forward primer: 5'-AGTTTGATCCTGGCTCA-3', reverse primer: 5'-TACGGCTACCTTGTTACGACTT-3'. The PCR product amplified to the expected size was submitted to Sangon Biotech (Shanghai) Co., Ltd. for purification and sequencing. Homology analysis of the sequenced 16S rRNA gene sequence of the strain was performed using the BLAST system in NCBI. The similarity to the published *Aeromonas hydrophila* was over 99%, further confirming that the strain was *Aeromonas hydrophila*.

[0039] The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on September 11, 2025, and classified and named as follows: Collection number: GDMCC No:66953, Address: 5th Floor, Building 59, No.100 Xianlie Middle Road, Guangzhou.

[0040] Example 2: Preparation and characterization of Aeromonas hydrophila vaccine 1. Preparation of Aeromonas hydrophila vaccine Prepare Aeromonas hydrophila vaccine according to the following steps: S1. Preparation of primary strains Inoculate the frozen Aeromonas hydrophila Ah-221212 strain into brain heart extract medium and incubate at 26-32℃ for 12-36 hours. Pick more than 5 typical single colonies, inoculate them into brain heart extract medium, and incubate at 26-32℃ for 6-24 hours. After the purity is verified, it is used as the primary strain. Store at 2-8℃ for no more than 7 days. Passage on the medium, and the number of passages should not exceed 5.

[0041] S2. Preparation of secondary strains Inoculate the primary strain with brain and heart extract culture medium and incubate at 26-32℃ with shaking at 200-400 rpm for at least 48 hours. After testing its purity, it is used as the secondary strain. Store at 2-8℃ for no more than 7 days.

[0042] S3. Preparation, inactivation, and disruption of the inoculum solution for seedling production S31. Inoculate the brain and heart extract culture medium with 6%-10% (v / v) of the secondary bacterial strain, and incubate at 26-32℃ with shaking at 200-400 rpm for at least 48 hours. After centrifugation at 5000 rpm for 15 minutes, resuspend in PBS, count with a hemocytometer, and dilute to 10⁻⁶. 9 CFU / mL available for use.

[0043] S32. Add 0.5% formaldehyde to the bacterial suspension to inactivate it for 48 hours. Spread it on brain and heart infusion medium. If no colony growth occurs after 48 hours, it proves that the inactivation was successful. After successfully inactivating the bacterial suspension, centrifuge at 5000 rpm for 10 minutes and resuspend it. Wash it three times with PBS. Use an ultrasonic cell disruptor to sonicate the prepared bacterial suspension at 100-400 watts for 10-60 minutes. Let the disrupted bacterial suspension (referred to as the disrupted bacterial suspension) stand and store it at 4℃.

[0044] S4. Emulsification Double-distilled water, medium-chain triglycerides, and Tween 20 were mixed at a volume fraction of 60% of the broken bacterial solution, 27% of the double-distilled water, 8% of the medium-chain triglycerides, and 5% of the Tween 20. The mixture was homogenized in a homogenizer for 1 hour to fully emulsify and obtain an emulsified broken bacterial solution.

[0045] S5. Coated vaccine S51. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a concentration of 10 mg / mL. Stir the solution on a constant temperature magnetic stirrer for 4-12 hours until the solution is clear to obtain a chitosan solution.

[0046] S52. Add emulsified and broken bacterial solution to chitosan solution at a ratio of 1:1, stir on a constant temperature magnetic stirrer for 1-6 hours to fully coat the vaccine and obtain vaccine solution; in the obtained vaccine solution, the vaccine particles are chitosan nanoparticles carrying broken Aeromonas hydrophila cells.

[0047] 2. Characterization of chitosan solution (obtained in S51), lysed bacterial culture (obtained in S4), and vaccine solution (obtained in S52). The particle size, zeta potential, and polydispersity index of chitosan solution (obtained in S51), lysed bacterial culture (obtained in S4), and vaccine solution (obtained in S52) were determined using a Malvern laser particle size analyzer. The results showed that the particle size of the lysed bacterial culture was 263.80 ± 22.1 nm, which decreased to 230.57 ± 13.8 nm after preparation as a vaccine solution. The zeta potential changed from -14.44 mV to 37.25 mV, as shown in Table 1. This indicates that the chitosan nanoparticles carrying lysed Aeromonas hydrophila cells were successfully prepared. The decrease in vaccine particle size proves that the chitosan tightly encapsulates the lysed bacterial culture. Meanwhile, the particle size distributions of the chitosan solution, lysed bacterial culture, and vaccine solution are shown in Table 1. Figure 3 As shown, the final vaccine solution exhibits uniform particle size, approximately 200 nm. Furthermore, the electron micrographs of the particles in the three solutions are shown below. Figure 4 As shown, (a) is the bacterial solution in S32 before inactivation and disruption, (b) is the disrupted bacterial solution in S4 after emulsification, and (c) is the vaccine solution in S52. From... Figure 4 (c) It can be seen that the successfully encapsulated chitosan nanovaccine is a nearly spherical body with a dark and dense inner core and a light-colored outer shell, with complete and clear boundaries.

[0048] Table 1. Characterization results of chitosan solution, lysed bacterial culture, and vaccine solution.

[0049] Example 3: Safety Experiment 1.1 Selection and Temporary Holding of Experimental Fish One hundred and forty 3-month-old Yangtze sturgeon of similar size, with a body length of 14-16 cm and a weight of 9-12 g, were selected and divided into four groups. Two groups served as blank controls for immersion immunization and injection immunization, respectively, while the other two groups served as vaccine groups for immersion immunization and injection immunization. The sturgeon were temporarily raised in flowing water ponds for seven days.

[0050] 1.2 Safety Experiment Immersion group: 10L of water was added to a bucket, along with 100mL of the prepared vaccine solution (the vaccine stock solution prepared in Example 2 above). The Yangtze sturgeon in the immersion vaccine group were immersed in the water for 3 minutes for immunization. The control group was only immersed in the same amount of water. Mortality was observed in both groups of Yangtze sturgeon over 28 days. The results showed that no obvious pathological changes occurred in either group of Yangtze sturgeon within 28 days. The survival rate was 100% in both the control and vaccine groups, proving the safety of the vaccine.

[0051] Injection group: The vaccine solution (the vaccine stock solution prepared in Example 2 above) was diluted in PBS at a ratio of 1:100, and 200 μL was injected intraperitoneally into the fish. The control group was injected with only an equal volume of PBS. The mortality of the two groups of Yangtze sturgeon was observed within 28 days. The experimental results showed that no obvious pathological changes were observed in the two groups of Yangtze sturgeon within 28 days. The survival rate of the control group was 100%, and the survival rate of the vaccine group was 100%, proving that the vaccine is safe.

[0052] Example 4: Immunopotency Experiment of Vaccine (Aeromonas hydrophila Ah-221212 strain) 1. Materials and Methods (1) Vaccine Vaccine solution prepared in Example 2 (2) Efficacy testing strain Aeromonas hydrophila strain Ah-221212 was used for challenge experiments. (3) Laboratory animals The 3-month-old Yangtze sturgeon provided by the Chinese Sturgeon Research Institute in Yichang City, Hubei Province, are of similar size, with a body length of 14-16cm and a weight of 9-12g.

[0053] (4) Experimental design The experimental fish were divided into four groups: two blank control groups (immersion immunization and injection immunization) and two vaccine groups (immersion immunization and injection immunization). They were temporarily housed in a circulating aquaculture system for 7 days. 10L of water was added to a tank, along with 100mL of the prepared vaccine solution (the vaccine stock solution directly prepared in Example 2 above). The Yangtze sturgeon in the immersion vaccine group were immersed in the water for 3 minutes for immunization; the control group was only immersed in the same amount of water. The vaccine solution (the vaccine stock solution directly prepared in Example 2 above) was diluted with PBS at a ratio of 1:100, and 200μL was injected intraperitoneally into the injection group fish; the control group was only injected with the same amount of PBS.

[0054] 28 days after immunization, administer a median lethal dose (3 x 10⁻⁶). 6 An injection challenge experiment was conducted using Aeromonas hydrophila strain Ah-221212 (non-inactivated) at cfu / mL. The mortality rate of four groups of Yangtze sturgeon was observed within 14 days after challenge, and the relative protection rate was calculated.

[0055] 2. Results After immunization with the vaccine solution provided by this invention for 28 days, a challenge protection experiment was conducted on all immunized Yangtze sturgeon, including a blank control. Disease incidence and mortality were observed after challenge, and the protection rate of each batch of vaccine was calculated. Symptoms appearing after challenge were typical of sepsis, such as acute death, organ necrosis, and hemorrhage.

[0056] The results are shown in Table 2. As can be seen from the data in Table 2, approximately 70% of the Yangtze sturgeon in the non-immunized control group developed symptoms and approximately 50% died within 14 days after the challenge, confirming the success of the challenge experiment. The survival rate of the Yangtze sturgeon in the vaccine immersion group was 87.50%, with a relative protection rate of 77.78%; the survival rate of the Yangtze sturgeon in the vaccine injection group was 87.50%, with a relative protection rate of 75.00%. This demonstrates that the vaccine provided by this invention can effectively treat or prevent Aeromonas hydrophila infection, thus reducing the incidence of septicemia and other diseases caused by Aeromonas hydrophila. This has significant practical implications for promoting the further development of aquaculture and ecological balance, particularly for Yangtze sturgeon.

[0057] Table 2. Results of the protective effect (28 days after immunization) of Aeromonas hydrophila Ah-221212 strain vaccine against Yangtze sturgeon.

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. Application of Aeromonas hydrophila strain Ah-221212 in the preparation of a vaccine for preventing septicemia of aquatic animals caused by Aeromonas hydrophila, characterized in that: the preservation number of the Aeromonas hydrophila strain Ah-221212 is GDMCC No: 66953, the preservation address is Guangdong Microbial Culture Collection Center, and the preservation date is September 11, 2025.

2. The application of Aeromonas hydrophila strain Ah-221212 in the preparation of a vaccine for preventing septicemia of aquatic animals caused by Aeromonas hydrophila according to claim 1, characterized in that: the use object of the vaccine includes aquatic animals, and the aquatic animals include sturgeons.

3. The application of Aeromonas hydrophila strain Ah-221212 in the preparation of a vaccine for preventing septicemia of aquatic animals caused by Aeromonas hydrophila according to claim 1, characterized in that: the use object of the vaccine is Yangtze River sturgeon. The preparation method of the vaccine is as follows: S1. Prepare a primary strain using the Aeromonas hydrophila strain Ah-221212; S2. Prepare a secondary strain using the primary strain; 4. The use of Aeromonas hydrophila strain Ah-221212 according to claim 1 for the preparation of a vaccine for the prevention of septicemia in aquatic animals caused by Aeromonas hydrophila, characterized in that, S4. Emulsify the broken bacterial liquid obtained after S3 to obtain an emulsified broken bacterial liquid; S5. Prepare a chitosan solution, mix the chitosan solution with the emulsified broken bacterial liquid to coat the broken bacterial bodies, and obtain the vaccine. The specific operation of S1 is as follows: S3. The secondary was resuspended to 10 8 ~10 10 cfu / mL and the resulting bacterial solution was inactivated and sonicated; Take the frozen strain of Aeromonas hydrophila Ah-221212 and inoculate it into brain heart infusion medium, and cultivate it at 26-32°C for 12-36 hours. Typical single colonies are selected and inoculated into brain heart infusion medium, and cultivated at 26-32°C for 6-24 hours. After passing the purity test, the primary strain is obtained. The specific operation of S2 is as follows:

5. The use of Aeromonas hydrophila strain Ah-221212 according to claim 4 for the preparation of a vaccine for the prevention of septicemia in aquatic animals caused by Aeromonas hydrophila, characterized in that, Take the primary strain and inoculate it into brain heart infusion medium, and cultivate it at 26-32°C with 200-400 rpm shaking for more than 48 hours. After passing the purity test, the secondary strain is obtained. The specific operation of S3 is as follows:

6. The use of Aeromonas hydrophila strain Ah-221212 according to claim 4 for the preparation of a vaccine for the prevention of septicemia in aquatic animals caused by Aeromonas hydrophila, characterized in that, S32. Add 0.45-0.55% formaldehyde to the bacterial liquid prepared in S31 and inactivate it for 44-52 hours. After centrifugation at 4500-5500 rpm for 10-20 minutes, resuspend the inactivated bacterial liquid, and wash it with PBS for 2-4 times. Then, ultrasonically break the bacterial liquid for 10-60 minutes at a power of 100-400 watts. The specific operation of S4 is as follows:

7. The use of Aeromonas hydrophila strain Ah-221212 according to claim 4 for the preparation of a vaccine for the prevention of septicemia in aquatic animals caused by Aeromonas hydrophila, characterized in that, Mix and emulsify double distilled water, medium-chain triglyceride, Tween 20, and the broken bacterial liquid at a volume ratio of 50-70:22-32:6-10:4-6. S31. Take the secondary strain to inoculate brain heart infusion medium at a volume ratio of 6%-10%, and culture at 26-32°C and 200-400 rpm for not less than 48 hours. After centrifugation at 4500-5500 rpm for 10-20 minutes, resuspend with PBS, count with a hemocytometer, and dilute to 10 8 ~10 10 cfu / mL to obtain a seed liquid. S32. Take the seed liquid to inoculate brain heart infusion medium at a volume ratio of 6%-10%, and culture at 26-32°C and 200-400 rpm for not less than 48 hours. After centrifugation at 4500-5500 rpm for 10-20 minutes, resuspend with PBS, count with a hemocytometer, and dilute to 10 8 ~10 10 cfu / mL to obtain a seed liquid. S33. Take the seed In the vaccine, the average particle size of the vaccine particles is 230.57±13.8 nm.

8. The use of Aeromonas hydrophila strain Ah-221212 according to claim 4 for the preparation of a vaccine for the prevention of septicemia in aquatic animals caused by Aeromonas hydrophila, characterized in that, The specific operation of S5 is as follows: S51. Dissolve chitosan in an acetic acid solution with a volume fraction of 0.5-1.5% to prepare a chitosan solution with a concentration of 8-12 mg / mL; 9. The use of Aeromonas hydrophila strain Ah-221212 of claim 4 for the preparation of a vaccine for the prevention of septicemia in aquatic animals caused by Aeromonas hydrophila, characterized in that, S52. Add the emulsified broken bacterial liquid to the chitosan solution at a volume ratio of 1-1.5:1-1.5, and stir at a constant temperature for 1-6 hours to fully coat the broken bacterial bodies.

10. The use of Aeromonas hydrophila strain Ah-221212 according to claim 4 for the preparation of a vaccine for the prevention of septicemia in aquatic animals caused by Aeromonas hydrophila, characterized in that, ​ ​ ​

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