An a. hydrophila and a. veronii septicemia double inactivated vaccine and a preparation method thereof

Abstract

This invention relates to the field of biopharmaceutical technology, specifically to a bivalent inactivated vaccine against grass carp caused by *Aeromonas hydrophila* and *Aeromonas vesiculosus* septicemia, and its preparation method. The preparation method of the bivalent inactivated vaccine includes the following steps: inactivating *Aeromonas hydrophila* and *Aeromonas vesiculosus* vaccine preparation solutions separately, and then mixing them with sterile sodium chloride solution and an adjuvant. The bivalent inactivated vaccine prepared by this invention has high safety, no adjuvant residue risk, and a high relative protection rate.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a bivalent inactivated vaccine for grass carp containing Aeromonas hydrophila and Aeromonas verrucosa septicemia, and its preparation method. Background Technology

[0002] Grass carp, a major freshwater aquaculture species in my country, boasts a wide range of adaptable farming areas and high yields. According to 2024 aquaculture production statistics, the total national freshwater aquaculture output reached 35.32 million tons, of which grass carp accounted for 6.16 million tons, or 17.5% of the total, holding a vital position in my country's freshwater aquaculture industry. Aeromonas septicemia is a highly destructive bacterial disease affecting aquaculture species. It can infect various freshwater fish, including grass carp, crucian carp, silver carp, bighead carp, bluntnose bream, and mandarin fish, causing significant direct and indirect economic losses to the freshwater aquaculture industry annually. The disease peaks from June to September each year, exhibiting an acute and explosive course. Under normal disease conditions, fish mortality rates can reach 50%-60%, and in severe cases, mortality can exceed 80%.

[0003] Epidemiological data from the past decade or so confirms that bacterial septicemia has posed a serious threat to the large-scale grass carp farming industry and is a key disease factor restricting the healthy development of the grass carp farming industry. Epidemiological source tracing investigations show that this pathogen is mainly caused by infection with Aeromonas verrucosa and Aeromonas hydrophila. Among the pathogens isolated and identified from the internal organs of naturally infected fish, Aeromonas verrucosa accounted for the highest proportion, while Aeromonas hydrophila was the second most prevalent pathogen. Fish naturally infected with Aeromonas exhibit typical pathological features, with widespread congestion lesions on the body surface, especially prominent in the jaw, gill covers, fin bases, and abdomen; accompanied by redness and swelling of the anus and abdominal distension; squeezing the anus of diseased fish can expel yellow viscous body fluid; the gill tissue shows pathological changes such as pale gill filaments and congestion and swelling. Necropsy of diseased fish reveals pale yellow ascites in the abdominal cavity, edema of the intestinal tissue, and diffuse hemorrhage. In the early stages of the disease, the pathological features of the liver and kidneys are not obvious. As the disease progresses to the middle and late stages, the liver and kidneys will show severe anemia, swelling and whitish color, and the spleen will be abnormally enlarged and purplish-black. The damage to the fish's organs is irreversible, which greatly increases the probability of death of the diseased fish.

[0004] Currently, the high morbidity and mortality rates of bacterial Aeromonas septicemia severely restrict the green, efficient, and large-scale development of the grass carp farming industry. The industry lacks effective immunomodulatory control technologies for septicemia caused by this complex pathogen. Therefore, research on a bivalent inactivated vaccine against grass carp Aeromonas hydrophila and Aeromonas vesiculosus septicemia has significant practical importance and broad application prospects. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to obtain a bivalent inactivated vaccine against grass carp aeromonas hydrophila and aeromonas verrucosa sepsis, and its preparation method.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a method for preparing a bivalent inactivated vaccine against grass carp caused by *Aeromonas hydrophila* and *Aeromonas vesiculosus* septicemia, comprising the following steps: inactivating *Aeromonas hydrophila* and *Aeromonas vesiculosus* vaccine preparation solutions separately, and then mixing them with sterile sodium chloride solution and adjuvants to obtain the vaccine. The specific process is as follows: Figure 1 As shown, the Aeromonas hydrophila culture used for seed preparation was prepared from Aeromonas hydrophila AHCI16, which was deposited on June 4, 2026 at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 68420) at the Institute of Microbiology, Guangdong Academy of Sciences. Aeromonas veronii AVCI11 was also deposited on June 4, 2026 at the same center with accession number GDMCC No.: 68419.

[0007] In some embodiments, the method for preparing the Aeromonas hydrophila seed culture includes the following steps: A1. Preparation of seeds for production: Take the frozen Aeromonas hydrophila AHCI16 strain, inoculate it onto nutrient agar medium, and thaw and culture it at 25-30℃ for 18-24 hours; pick a single colony and streak it onto nutrient agar plates, and culture it at 25-30℃ for 18-24 hours. After passing the purity test, it is used as the primary seed. Take the primary seed and inoculate it onto nutrient broth medium, and culture it at 25-30℃ and 180-220 rpm for 12-14 hours. After passing the purity test, it is used as the secondary seed and stored at 2-8℃ for later use. The storage period is ≤5 days. A2. Preparation of bacterial culture: The culture was carried out in a fermenter. After sterilization, the Aeromonas hydrophila fermentation medium was inoculated with the secondary seed prepared in step A1 at 9-11% of the total volume of the medium. The culture was carried out at 25-30℃, with an aeration rate of 3.5-4.4 L / min and a stirring speed of 180-220 rpm for 10-12 hours to obtain the Aeromonas hydrophila seedling solution.

[0008] In some embodiments, the Aeromonas hydrophila fermentation medium comprises the following components in the following amounts: bacteriological peptone 8-15 g / L, glucose 2-7 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate 0.2-0.6 g / L, beef extract 1-5 g / L, and sodium chloride 3-8 g / L; the solvent is water, and the pH is 7-8.

[0009] Preferably, the Aeromonas hydrophila fermentation medium comprises the following components: 10.0 g / L bacteriological peptone, 5.0 g / L glucose, 1.5 g / L potassium dihydrogen phosphate, 0.40 g / L magnesium sulfate, 3.0 g / L beef extract, and 5.0 g / L sodium chloride; the solvent is water, and the pH is 7.5.

[0010] In some embodiments, the method for preparing the Aeromonas verrucosa vaccine culture includes the following steps: B1. Preparation of seeds for production: Take the frozen Aeromonas villi AVCI11 strain, inoculate it onto nutrient agar medium, and thaw and culture it at 25-30℃ for 18-24 hours; pick a single colony and streak it onto nutrient agar plates, and culture it at 25-30℃ for 18-24 hours. After passing the purity test, it is used as the primary seed. Take the primary seed and inoculate it onto nutrient broth medium, and culture it at 25-30℃ and 180-220 rpm for 12-14 hours. After passing the purity test, it is used as the secondary seed and stored at 2-8℃ for later use. The storage period is ≤5 days. B2. Preparation of bacterial culture: The culture was carried out in a fermenter. After sterilizing the Aeromonas versicolor fermentation medium, the secondary seed prepared in step B1 was inoculated at 9-11% of the total volume of the medium. The culture was carried out at 25-30℃, with an aeration rate of 3.5-4.4 L / min and a stirring speed of 180-220 rpm for 10-12 hours to obtain the Aeromonas versicolor seedling culture.

[0011] In some embodiments, the Aeromonas vera fermentation medium comprises the following components in the following amounts: tryptone 8-12 g / L, glucose 4-6 g / L, potassium dihydrogen phosphate 1-4 g / L, magnesium sulfate 0.2-0.6 g / L, beef extract 2-5 g / L, and sodium chloride 3-7 g / L; the solvent is water, and the pH is 7-8.

[0012] Preferably, the Aeromonas verrucosa fermentation medium contains the following components in the following amounts: tryptone 10.0 g / L, glucose 5.0 g / L, potassium dihydrogen phosphate 2.0 g / L, magnesium sulfate 0.40 g / L, beef extract 3.0 g / L, and sodium chloride 5.0 g / L; the solvent is water, and the pH is 7.5.

[0013] In some embodiments, the specific steps of inactivation are as follows: add formaldehyde solution to the bacterial solution, inactivate at 35-38℃ for 30-35 hours, and stir at 40-60 rpm for 3-5 minutes every 3-6 hours during this period.

[0014] In some embodiments, the final concentration of formaldehyde in the bacterial solution is 0.2-0.3% vol.

[0015] In some embodiments, the adjuvant is aluminum hydroxide gel.

[0016] The second aspect of this invention provides a bivalent inactivated vaccine against grass carp aeromonas hydrophila and aeromonas versicolor sepsis.

[0017] In some embodiments, the vaccine contains at least 2.0 × 10⁻⁶ viable counts of Aeromonas hydrophila and Aeromonas vesiculosus before inactivation. 9 CFU / ml.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The grass carp Aeromonas hydrophila and Aeromonas versicolor septicemia bivalent vaccine disclosed in this invention is an inactivated vaccine that retains immunogenicity, has high safety, mature preparation technology, no adjuvant residue risk, and high relative protection rate. Attached Figure Description

[0019] Figure 1 Flowchart for the preparation of a bivalent inactivated vaccine against septicemia caused by Aeromonas hydrophila and Aeromonas vernix in grass carp; Figure 2 The image shows the morphological identification results of Aeromonas hydrophila AHCI16. Figure 3 The image shows the biochemical reaction results of Aeromonas hydrophila AHCI16. Figure 4 The image shows the sequencing results of the 16S rRNA gene of Aeromonas hydrophila AHCI16. Figure 5 Image showing the sequencing results of the gyrB gene in Aeromonas hydrophila AHCI16; Figure 6 Phylogenetic tree diagram of Aeromonas hydrophila AHCI16; Figure 7 Figure A shows the results of the detection of Aeromonas hydrophila AHCI16, and Figure B shows the colony morphology. Figure 8 The image shows the morphological identification results of Aeromonas versicolor AVCI11. Figure 9 The diagram shows the biochemical reaction results of Aeromonas vulgaris AVCI11. Figure 10The image shows the sequencing results of the 16S rRNA gene of Aeromonas versicolor AVCI11. Figure 11 Image showing the sequencing results of the gyrB gene of Aeromonas versicolor AVCI11; Figure 12 Phylogenetic tree of Aeromonas versicolor AVCI11; Figure 13 The results of the detection of Aeromonas verrucosa AVCI11 are shown in Figure A, which is a colony morphology diagram, and Figure B is a microscopic morphology diagram. Figure 14 The image shows the anatomical observation results of fish challenged with Aeromonas hydrophila AHCI16. In the image, AB represents the dead fish in the intraperitoneal injection control group, CD represents the dead fish in the intraperitoneal injection immunization group, and EF represents the healthy fish in the intraperitoneal injection immunization group. Figure 15 Figure 1 shows the anatomical observation results of fish challenged with Aeromonas hydrophila AHCI16. Figure 2A and 3B are fish that died in the intramuscular injection control group, Figure 3C and 4D are fish that died in the intramuscular injection immunization group, and Figure 5E and 6E are healthy fish in the intramuscular injection immunization group. Figure 16 The images show the anatomical observation results of fish challenged with Aeromonas vulgaris AVCI11. In the images, AB represents the dead fish in the intraperitoneal injection control group, CD represents the dead fish in the intraperitoneal injection immunization group, and EF represents the healthy fish in the intraperitoneal injection immunization group. Figure 17 Figures show the anatomical observation results of fish challenged with Aeromonas vulgaris AVCI11. Figures AB show the dead fish in the intramuscular injection control group, Figures CD show the dead fish in the intramuscular injection immunization group, and Figures EF show the healthy fish in the intramuscular injection immunization group. Detailed Implementation

[0020] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0021] In the following examples and comparative examples, the compounds and related reagents used were all commercially available. Nutrient agar, bacteriological peptone, tryptone, and beef extract were purchased from Guangdong Huankai Microbial Technology Co., Ltd.; aluminum hydroxide gel was purchased from Ruimeng Company, USA.

[0022] Unless otherwise specified, the post-processing steps used below are routine operations for those skilled in the art, and can be selected according to actual operations; pure testing shall be carried out in accordance with the appendix of Part III of the current Chinese Veterinary Pharmacopoeia.

[0023] Preparation Example 1: Isolation and Identification of Aeromonas hydrophila 1. Strains Aeromonas hydrophila was isolated from diseased grass carp in a fish farm in a reservoir in Shaoguan City, Guangdong Province.

[0024] 2. Methods 2.1 Screening of vaccine strains The diseased grass carp were collected from a fish farm in Shaoguan City, Guangdong Province. The main symptoms of the disease were widespread congestion on the body surface, particularly noticeable in the jaw, gill covers, fin bases, and abdomen. Other symptoms included redness and swelling of the anus, abdominal swelling, and the discharge of a thick, yellowish fluid upon squeezing the anus. The gills were pale or congested and swollen. Post-mortem examination revealed pale yellow ascites in most cases, edematous intestines, and diffuse hemorrhage. The diseased grass carp were rinsed three times with physiological saline, and liver tissue was harvested under aseptic conditions. After rinsing the tissue blocks with physiological saline, they were placed in sterile 2.0 ml centrifuge tubes, chopped, and a small amount was streaked onto a nutrient agar plate and incubated at 28°C for 24 hours. Once colonies had grown, single colonies of uniform size and color were selected for identification.

[0025] 2.2 Identification Results: 1. Morphological identification results: The colonies on nutrient agar medium are round, moist, smooth, with neat edges, and pale yellow in color. Figure 2 A); Blood agar culture medium exhibits a typical transparent hemolytic zone (A); Figure 2 B). Gram staining and microscopic observation revealed them to be Gram-negative short bacilli, mostly arranged singly. Figure 2 C).

[0026] 2. Test Results like Figure 3 AHCI16 biochemical reaction results diagram Figure 4 AHCI16 16S rRNA gene sequencing results image. Figure 5 The sequencing results of the AHCI16 gyrB gene are shown in the figure.

[0027] Based on the sequencing results above, sequence alignment analysis was performed, and a phylogenetic tree was constructed based on the alignment results, as follows: Figure 6 As shown, Aeromonas hydrophila was named Aeromonas hydrophila AHCI16, as... Figure 7 As shown.

[0028] Preparation Example 2: Isolation and Identification of Aeromonas versicolor 1. Strains Aeromonas villus was isolated from diseased grass carp at a fish farm in Nansha, Guangdong Province.

[0029] 2. Methods 2.1 Screening of vaccine strains The diseased grass carp were collected from a fish farm in Nansha, Guangdong Province. The main symptoms were widespread congestion on the body surface, particularly noticeable in the jaw, gill covers, fin bases, and abdomen. Other signs included redness and swelling of the anus, abdominal swelling, and the discharge of a thick, yellowish fluid upon squeezing the anus. The gills were pale or congested and swollen. Post-mortem examination revealed pale yellow ascites, edematous intestines, and diffuse hemorrhage in most cases. The diseased grass carp were rinsed three times with physiological saline, and liver tissue was harvested under aseptic conditions. After rinsing the tissue blocks with physiological saline, they were placed in sterile 2.0 ml centrifuge tubes, chopped, and a small amount was streaked onto a nutrient agar plate and incubated at 28°C for 24 hours. Once colonies had grown, single colonies of uniform size and color were selected for identification.

[0030] 2.2 Identification Results: 1. Morphological identification results: The strain formed round, well-defined, smooth, pale yellow, raised colonies after 24 hours of culture on nutrient agar plates. Figure 8 A), hemolysis was observed on blood agar medium. Figure 8 B). Gram-negative; bacteria are short rod-shaped or arc-shaped with blunt ends (B). Figure 8 C).

[0031] 2. Test Results like Figure 9 AVCI11 biochemical reaction results diagram, Figure 10 Image of AVCI11 16S rRNA gene sequencing results. Figure 11 The sequencing results of the AVCI11 gyrB gene are shown in the figure.

[0032] Based on the sequencing results above, the sequences were compared and analyzed, and a phylogenetic tree was constructed based on the comparison results as follows: Figure 12 As shown, Aeromonas vesiculosus was also named Aeromonas vesiculosus AVCI11, as... Figure 13 As shown.

[0033] Preparation Example 3 The method for preparing bacterial suspension for Aeromonas hydrophila seed production includes the following steps: A1. Preparation of seeds for production: Take the frozen strain of Aeromonas hydrophila AHCI16, inoculate it on nutrient agar medium, and thaw and culture it at 28℃ for 22h. Pick a single colony and streak it onto nutrient agar plates, and culture it at 28℃ for 22h. After passing the purity test, it is used as the primary seed. Take the primary seed and inoculate it on nutrient broth medium at an inoculation rate of 2% vol of the total culture medium volume. Culture it at 28℃ and 200 rpm for 13h. After passing the purity test, it is used as the secondary seed. Store the strain at 3℃ for later use. The storage period is ≤5 days. Nutrient broth medium: bacteriological peptone 10.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, agar 18.0 g / L, solvent: deionized water, pH=7.5; A2. Preparation of bacterial culture: A 7L fermenter was used for culture. After sterilizing the Aeromonas hydrophila fermentation medium, secondary seeds were inoculated at 10% of the total volume of the medium. The culture was carried out at 28℃, with an aeration rate of 4L / min and a stirring speed of 200rpm for 11h to obtain the Aeromonas hydrophila seedling solution. The Aeromonas hydrophila fermentation medium contains the following components: bacteriological peptone 10.0 g / L, glucose 5.0 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.40 g / L, beef extract 3.0 g / L, and sodium chloride 5.0 g / L; the solvent is deionized water, pH=7.5.

[0034] Preparation Example 4 The method for preparing Aeromonas verrucosa bacterial suspension for vaccine production includes the following steps: B1. Preparation of seeds for production: Take the frozen Aeromonas villi AVCI11 strain, inoculate it onto nutrient agar medium, and thaw and culture at 28℃ for 22 h; pick a single colony and streak it onto nutrient agar plates, culture at 28℃ for 22 h. After passing the purity test, it is used as the primary seed. Take the primary seed and inoculate it onto nutrient broth medium at an inoculation rate of 2% vol of the total culture medium volume, culture at 28℃ and 200 rpm for 13 h. After passing the purity test, it is used as the secondary seed. Store the strain at 3℃ for later use, with a storage period of ≤5 days. Nutrient broth medium: bacteriological peptone 10.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, agar 18.0 g / L; solvent: deionized water, pH=7.5; B2. Preparation of bacterial culture: A 7L fermenter was used for culture. After sterilizing the Aeromonas versicolor fermentation medium, the secondary seed prepared in step B1 was inoculated at 10% of the total volume of the medium. The culture was carried out at 28℃, with an aeration rate of 4L / min and a stirring speed of 200rpm for 11h to obtain Aeromonas versicolor seedling culture. Aeromonas verrucosa fermentation medium contains the following components: 10.0 g / L tryptone, 5.0 g / L glucose, 2.0 g / L potassium dihydrogen phosphate, 0.40 g / L magnesium sulfate, 3.0 g / L beef extract, and 5.0 g / L sodium chloride; the solvent is deionized water, pH=7.5.

[0035] Three batches of Aeromonas hydrophila and Aeromonas vernix casei cultures were prepared in parallel according to the methods of Preparation Example 3 and Preparation Example 4. The viable cell counts of the three batches of cultures are shown in Table 1. Table 1

[0036] Example 1 A method for preparing a bivalent inactivated vaccine against grass carp Aeromonas hydrophila and Aeromonas vesiculosus septicemia includes the following steps: 2180 ml of Aeromonas hydrophila vaccine preparation solution 01001 and 2390 ml of Aeromonas vesiculosus vaccine preparation solution 02001 are added to formaldehyde solution to a final formaldehyde concentration of 0.25% vol. The mixture is inactivated at 37°C for 32 hours, with stirring at 50 rpm for 4 minutes every 4 hours. Then, the inactivated Aeromonas hydrophila vaccine preparation solution 01001 and Aeromonas vesiculosus vaccine preparation solution 02001 are mixed with 4430 ml of 0.65% vol sterile sodium chloride solution and 1000 ml of aluminum hydroxide gel.

[0037] Example 2 A method for preparing a bivalent inactivated vaccine against grass carp Aeromonas hydrophila and Aeromonas vesiculosus septicemia includes the following steps: 2050 ml of Aeromonas hydrophila vaccine preparation solution 01002 batch and 2360 ml of Aeromonas vesiculosus vaccine preparation solution 02002 batch are respectively added to formaldehyde solution to achieve a final formaldehyde concentration of 0.25% vol. The mixture is inactivated at 37°C for 32 hours, with stirring at 50 rpm for 4 minutes every 4 hours. Then, the inactivated Aeromonas hydrophila vaccine preparation solution 01002 batch and Aeromonas vesiculosus vaccine preparation solution 02002 batch are mixed with 4590 ml of 0.65% vol sterile sodium chloride solution and 1000 ml of aluminum hydroxide gel.

[0038] Example 3 A method for preparing a bivalent inactivated vaccine against grass carp Aeromonas hydrophila and Aeromonas vesiculosus septicemia includes the following steps: 2090 ml of Aeromonas hydrophila vaccine preparation solution 01003 batch and 2470 ml of Aeromonas vesiculosus vaccine preparation solution 02003 batch are added to formaldehyde solution to a final formaldehyde concentration of 0.25% vol. The mixture is inactivated at 37°C for 32 hours, with stirring at 50 rpm for 4 minutes every 4 hours. Then, the inactivated Aeromonas hydrophila vaccine preparation solution 01003 batch and Aeromonas vesiculosus vaccine preparation solution 02003 batch are mixed with 4440 ml of 0.65% vol sterile sodium chloride solution and 1000 ml of aluminum hydroxide gel.

[0039] Performance testing The following tests were performed on the grass carp Aeromonas hydrophila and Aeromonas vesiculosus septicemia bivalent inactivated vaccines obtained in the above embodiments: I. Security 1. Safety Experiment 1.1 Experimental Vaccine The grass carp Aeromonas hydrophila and Aeromonas versicolor septicemia bivalent inactivated vaccine was prepared as described in Examples 1-3.

[0040] 1.2 Experimental Animals The minimum healthy grass carp size for use is 10-20g. They are from the Guangzhou Fangcun Experimental Base of the Pearl River Fisheries Research Institute. They are randomly selected according to the criteria for healthy grass carp and used for the experiment. One week before the experiment, they are moved to the aquatic animal house of the Pearl River Fisheries Research Institute for aquarium rearing. Two days before vaccination, the physical condition of the experimental fish is further verified. The criteria for judging healthy grass carp are: (1) The fish swim normally, in groups, and with vigorous movement. They are sensitive to fright and do not exhibit any abnormalities such as slow or rotating movements. (2) The fish have normal body color, appearing bluish-gray without any abnormal symptoms such as darkening or blackening. (3) The fish are intact, with scales tightly attached to the skin, intact without any falling off or standing up. There is mucus on the body surface, no bleeding or lumps, and all fins are intact. There are no signs of disease such as red mouth, red spots, rotten tail, rotten edges, white spots, swelling, or congestion. (4) The fish feed normally without slow or no feeding. (5) There is no white feces or dragging feces. (6) Before the experiment, a certain number of grass carp (less than 100 fish) were randomly selected from the experimental fish group. Take 5 fish, randomly select 10 fish from a group of 100-500 fish, and randomly select 20 fish from a group of more than 500 fish. Observe that the gills are intact, bright red, and without silt or bacterial clumps. Take samples from the body surface and gills, make smears, and observe under a microscope. There should be no parasitic infection. After dissecting the fish, there should be no ascites. The color of the muscles, liver, spleen, kidneys, gallbladder, intestines and other organs should be normal, without swelling or bleeding. Use nutrient agar plates to isolate bacteria from the liver, spleen and kidneys. There should be no bacterial growth. (7) Test the grass carp according to the grass carp reovirus type II PCR detection method. The test result should be negative.

[0041] 2 methods 2.1 Grouping and Identification Prepare healthy grass carp weighing 10-20g for testing, randomly divide them into 8 groups of 50 fish each, and keep them in different aquariums of the same size.

[0042] 2.2 Immunization Groups 1-3 (experimental groups) received intraperitoneal injections of the vaccine prepared in Examples 1-3, with the needle inserted obliquely 0.2 cm from the base of the pelvic fin of the experimental fish, and 0.2 ml of vaccine per fish. Group 4 (control group) received intraperitoneal injections of sterile 0.65% vol sodium chloride solution, 0.2 ml per fish. Groups 5-7 (experimental groups) received intramuscular injections of the vaccine prepared in Examples 1-3, with the needle inserted obliquely 0.2 cm from the base of the dorsal fin of the experimental fish, and 0.2 ml per fish. Group 8 (control group) received intramuscular injections of sterile 0.65% vol sodium chloride solution, 0.2 ml per fish. Observations were conducted for 14 consecutive days.

[0043] 2.3 Experimental Fish Rearing 2.3.1 Management Every day, drain the bottom sludge and add a small amount of clean water to maintain the water level and keep the water temperature between 27 and 29 degrees Celsius.

[0044] 2.3.2 Feeding Feed the fish an appropriate amount of pelleted feed at 9 am and 5 pm each day, with the daily feeding amount being about 3% of the total weight of the fish.

[0045] 2.4 Clinical observation 2.4.1 Daily Observation Observe and record changes in water temperature, feeding, and swimming patterns of the experimental fish daily, and count the number of dead and surviving fish. If grass carp die during the observation period, analyze and determine whether it is a non-specific death according to the "Criteria for Determining Non-Specific Deaths in Grass Carp".

[0046] 2.4.2 Anatomical observation Fourteen days after immunization, five healthy grass carp were randomly dissected to observe the body surface, gills, liver, spleen, kidneys and other tissues and organs of the experimental group fish. The results were compared with those of the control group to see if any abnormalities were found.

[0047] 3 Results 3.1 Routine clinical observation Healthy grass carp were injected with a single dose of vaccine (0.2 ml / fish) via intraperitoneal or intramuscular injection. They were observed for 14 consecutive days after vaccination. The results showed that no abnormal clinical symptoms were observed in either the intraperitoneal injection experimental fish or the control fish. Some of the intramuscular injection experimental fish and the control fish developed ecchymosis at the injection site, and the wounds healed within 3 days. Throughout the observation period, the grass carp in each experimental group quickly floated to the surface to feed when fed, and their swimming behavior was no different from that of the control group. No abnormalities were observed in body color, feces, etc.

[0048] 3.2 Anatomical observation Fourteen days after a single dose of inoculation, no abnormal changes were observed on the fish's body surface, oral cavity, and gills; upon necropsy, no boils or furuncles were found at the injection site; there was no ascites in the abdominal cavity; and no lesions such as congestion or hemorrhage were found in organs such as the liver, spleen, kidneys, and intestines; no abnormalities were found compared with the control fish.

[0049] During the safety observation period of a single-dose repeated vaccination of grass carp with a bivalent inactivated vaccine against Aeromonas hydrophila and Aeromonas vesiculosus septicemia, no abnormal clinical symptoms were observed in either the intraperitoneal injection experimental fish or the control fish. In some intramuscular injection experimental fish and control fish, ecchymosis appeared at the injection site, but the wounds healed within 3 days. Grass carp in all experimental groups rapidly floated to the surface to feed, exhibiting no difference in swimming activity compared to the control group, and no abnormalities were observed in body color or feces. No boils or abscesses were found at the injection site during necropsy; no lesions were found in the tissues and organs of the injected fish. The results indicate that the single-dose repeated vaccination of grass carp with this vaccine has good safety.

[0050] II. Adjuvant Residue Detection Test in Target Animals 1. Materials 1.1 Experimental Vaccine The experimental vaccine was the grass carp Aeromonas hydrophila and Aeromonas versicolor septicemia bivalent inactivated vaccine prepared in Example 1.

[0051] 1.2 Experimental Animals Grass carp weighing 10-20g were sourced from the Guangzhou Fangcun Experimental Base of the Pearl River Fisheries Research Institute. Random sampling was conducted according to the criteria for healthy grass carp, and qualified grass carp were selected for the experiment. One week before the experiment, the fish were transferred to aquariums in the aquatic animal house of the Pearl River Fisheries Research Institute for rearing. Two days before vaccination, the physical condition of the experimental fish was further verified.

[0052] 2 Methods 2.1 Grouping and Identification Prepare healthy grass carp weighing 10-20g for the experiment, randomly group them into groups of 50 fish, and keep them in different aquariums of the same size.

[0053] 2.2 Immunization The experimental group received intraperitoneal injection of the vaccine from Example 1, with the needle inserted obliquely 0.2 cm from the base of the pelvic fin of the experimental fish, and 0.2 ml per fish. The control group received intraperitoneal injection of sterile 0.65% vol sodium chloride solution, 0.2 ml per fish.

[0054] 2.3 Experimental Fish Rearing 2.3.1 Management: Drain the bottom sludge daily, add a small amount of clean water to maintain the water level, and control the water temperature at 27~29℃.

[0055] 2.3.2 Feeding: Feed an appropriate amount of pelleted feed at 9:00 AM and 5:00 PM every day.

[0056] 2.4 Clinical observation 2.4.1 Daily Observation Observe and record daily changes in water temperature, water quality, fish feeding and swimming, and changes in injection wounds in the experimental pond, and count the number of dead and surviving fish. If grass carp are found to have died during the observation period, they should be immediately removed and dissected, and analyzed according to the "Criteria for Determining Non-Specific Deaths in Grass Carp" to determine whether the death was non-specific.

[0057] 2.4.2 Adjuvant Residue Detection Three experimental fish were randomly selected from the control group and the experimental groups on days 3, 7, 14, 30 and 90 after inoculation for dissection. Five g of muscle tissue was taken from each fish. The residual amount of aluminum was determined by graphite furnace atomic absorption spectrometry according to the determination method in GB5009.182-2017 Determination of Aluminum in Food.

[0058] 3 Results 3.1 Routine clinical observation Grass carp were injected intraperitoneally with a single dose of vaccine (0.2 ml / fish). They were observed for 14 consecutive days after vaccination. The results showed that no abnormal clinical symptoms appeared in any of the fish during the entire observation period. Grass carp in the experimental group floated to the surface to feed quickly when fed, and their swimming behavior was no different from that of the control group. No abnormalities were observed in body color, feces, etc.

[0059] 3.2 Adjuvant Residue Detection Three fish were randomly sampled from the control group and the experimental groups on days 3, 7, 14, 30 and 90 after inoculation (labeled as experimental groups D3, D7, D14, D30 and D90, respectively). The residual amount of aluminum in the muscle was detected to characterize the residual amount of aluminum adjuvant (unit: mg). The results are shown in Table 2.

[0060] Table 2

[0061] Healthy grass carp were intraperitoneally injected with a single dose of the bivalent inactivated vaccine against Aeromonas hydrophila and Aeromonas vesiculoskeletal septicemia. During the observation period, neither the experimental nor control fish exhibited any abnormal clinical symptoms. The aluminum content in the muscle tissue of the experimental and control groups was measured. Results showed that on days 3, 7, 14, 30, and 90 post-immunization, the average aluminum content per gram of muscle tissue in the experimental group was 3.2 mg, 1.1 mg, 1.2 mg, 3.3 mg, and 1.2 mg, respectively. Compared to the control group (aluminum content of 1.4 mg per gram of muscle tissue), the aluminum content in the muscle tissue did not increase significantly during the experimental period. Therefore, the residual aluminum adjuvant had no impact on the commercial value or food safety of grass carp. Based on this, it can be concluded that there is no withdrawal period for grass carp after injection of the bivalent inactivated vaccine against Aeromonas hydrophila and Aeromonas vesiculoskeletal septicemia.

[0062] III. Immunopotency Test 1. Materials 1.1 Experimental Vaccine The experimental vaccine was the grass carp Aeromonas hydrophila and Aeromonas versicolor septicemia bivalent inactivated vaccine prepared in Examples 1-3.

[0063] 1.2 Experimental Animals 10-20g of healthy grass carp were obtained from the Guangzhou Fangcun Experimental Base of the Pearl River Fisheries Research Institute. Random sampling was conducted according to the criteria for healthy grass carp, and qualified grass carp were selected for the experiment. One week prior to the experiment, the fish were moved to aquariums in the aquatic animal facility of the Pearl River Fisheries Research Institute. Two days before vaccination, the health status of the experimental fish was further verified.

[0064] 1.3 Test strains Aeromonas hydrophila AHCI16 and Aeromonas vernix AVCI11.

[0065] 2 Methods 2.1 Grouping and Identification Healthy grass carp weighing 10-20g were selected and randomly divided into 8 groups of 120 fish each, and kept in different aquariums of the same size.

[0066] 2.2 Immunization Intraperitoneal immunization: Groups 1-3 were injected intraperitoneally with the vaccines prepared in Examples 1-3, with the needle inserted obliquely 0.2 cm from the base of the pelvic fin of the experimental fish, and 0.2 ml of vaccine was injected per fish. Group 4 was injected intraperitoneally with sterile 0.65% vol sodium chloride solution, 0.2 ml per fish.

[0067] For intramuscular immunization, groups 5-7 were injected intramuscularly with the vaccine prepared in Examples 1-3, with the needle inserted obliquely at the base of the dorsal fin of the experimental fish at a depth of 0.2 cm, 0.2 ml / fish. Group 8 was injected intramuscularly with sterile 0.65% vol sodium chloride solution, 0.2 ml / fish.

[0068] 2.3 Experimental Fish Rearing 2.3.1 Management: Drain the bottom sludge daily, add a small amount of clean water to maintain the water level, and control the water temperature at 27~29℃.

[0069] 2.3.2 Feeding: Feed an appropriate amount of pelleted feed at 9:00 AM and 5:00 PM every day.

[0070] 2.4 Attacking the virus 2.4.1 Aeromonas hydrophila section Twenty-one days after immunization, 50 healthy grass carp were randomly selected from each experimental group and control group, and each fish was intraperitoneally injected with 0.2 ml / fish of Aeromonas hydrophila strain AHCI16 (containing approximately 8.0 × 10⁻⁶ live bacteria). 6(CFU / tail), withheld from food for 24 hours before and 72 hours after the challenge. Each group was housed in water at 27-29℃ and observed for 7 days. The number of dead fish in each group was recorded daily. Grass carp were considered healthy when they swam normally, had normal body color, were intact, and were feeding normally. The relative protection rate was calculated using the following formula: Mortality rate = (Number of dead fish per group / Total number of fish per group) × 100%.

[0071] Relative protection rate (%) = ×100% 2.4.2 Aeromonas versicolor (partial) Twenty-one days after immunization, 50 healthy grass carp were randomly selected from each experimental group and control group, and each fish was intraperitoneally injected with 0.2 ml / fish of Aeromonas vera AVCI11 virulent strain (containing approximately 6.0 × 10⁻⁶ live bacteria). 6 (CFU / tail), fasted for 24h and 72h before and after the challenge, respectively. Each group was kept in water at 27~29℃ and observed for 7 days. The number of dead fish in each group was recorded daily. When the experimental fish had normal swimming posture, normal body color, intact body, and normal feeding, they were judged to be healthy. The relative protection rate of the experimental group was calculated according to the above formula.

[0072] 2.5 Clinical Observation 2.5.1 Daily Observation Observe and record changes in water temperature, feeding, and swimming patterns of the experimental fish daily, and count the number of dead and surviving fish. If grass carp die during the observation period, analyze and determine whether it is a non-specific death according to the "Criteria for Determining Non-Specific Deaths in Grass Carp".

[0073] 2.5.2 Anatomical observation After the experimental fish were challenged with the virus, healthy and dead grass carp were randomly selected from each group for dissection. The tissues and organs of the experimental fish, such as the body surface, gills, liver, spleen, and kidneys, were observed and the results were recorded.

[0074] 3 Results 3.1 Results of challenge with Aeromonas hydrophila AHCI16 3.1.1 Intraperitoneal injection of vaccine After injecting the vaccines prepared in Examples 1-3 into healthy grass carp intraperitoneally for 21 days, the experimental group and the control group were challenged by intraperitoneal injection of Aeromonas hydrophila AHCI16 bacterial solution. The results are shown in Table 3.

[0075] Table 3

[0076] 3.1.2 Intramuscular injection of vaccine After intramuscular injection of the vaccines prepared in Examples 1-3 into healthy grass carp for 21 days, the experimental group and the control group were challenged by intramuscular injection of Aeromonas hydrophila AHCI16 bacterial solution. The results are shown in Table 4.

[0077] Table 4

[0078] 3.2 Results of challenge with Aeromonas versicolor AVCI11 3.2.1 Intraperitoneal injection of vaccine After intraperitoneal injection of the vaccines prepared in Examples 1-3 into healthy grass carp for 21 days, the experimental group and the control group were challenged by intraperitoneal injection of Aeromonas verrucosa AVCI11. The results are shown in Table 5.

[0079] Table 5

[0080] 3.2.2 Vaccination via intramuscular injection After intramuscular injection of the vaccines prepared in Examples 1-3 into healthy grass carp for 21 days, the experimental group and the control group were challenged by intramuscular injection of Aeromonas verrucosa AVCI11. The results are shown in Table 6.

[0081] Table 6

[0082] 3.3 Clinical observation After injecting the vaccine into grass carp via intraperitoneal and intramuscular injections, the experimental and control fish were observed for 21 consecutive days. No abnormal clinical symptoms were observed in either fish. The fish quickly floated to the surface to feed when fed, and no abnormalities were observed in their swimming, body color, or feces.

[0083] Within 72 hours of challenge with Aeromonas hydrophila AHCI16, a large number of fish in the control group died, while a small number of fish in the experimental group also died. After 72 hours, no fish in the experimental group died. Dissection revealed that the dead fish in the control group had hemorrhage at the base of their fins and muscles, and ascites; the dead fish in the experimental group had surface hemorrhage and occasionally ascites; the healthy fish showed no abnormalities (see...). Figure 14 , Figure 15 After challenge with Aeromonas verrucosa AVCI11, the morbidity and mortality of the experimental fish were similar (see...). Figure 16 , Figure 17 ).

[0084] Experiments showed that intraperitoneal and intramuscular injection of the bivalent vaccine into healthy grass carp provided good immunoprotection against challenge with Aeromonas hydrophila AHCI16 and Aeromonas vernix AVCI11.

[0085] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a bivalent inactivated vaccine against grass carp Aeromonas hydrophila and Aeromonas vesiculosus septicemia, characterized in that, The method includes the following steps: Aeromonas hydrophila and Aeromonas vesiculosus culture for seed preparation are inactivated separately, and then mixed with sterile sodium chloride solution and adjuvant. The Aeromonas hydrophila culture for seed preparation is prepared from Aeromonas hydrophila AHCI16, and the Aeromonas vesiculosus culture for seed preparation is prepared from Aeromonas vesiculosus AVCI11. Aeromonas hydrophila AHCI16 was deposited on June 4, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No.: 68420), located at the Institute of Microbiology, Guangdong Academy of Sciences. Aeromonas vesiculosus AVCI11 was deposited on June 4, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No.: 68419), located at the Institute of Microbiology, Guangdong Academy of Sciences.

2. The method for preparing the grass carp Aeromonas hydrophila and Aeromonas vesiculosus bivalent inactivated vaccine for septicemia according to claim 1, characterized in that, The method for preparing the bacterial culture for Aeromonas hydrophila seed production includes the following steps: A1. Preparation of seeds for production: Take the frozen Aeromonas hydrophila AHCI16 strain, inoculate it onto nutrient agar medium, and thaw and culture it at 25-30℃ for 18-24 hours; pick a single colony and streak it onto nutrient agar plates, and culture it at 25-30℃ for 18-24 hours. After passing the purity test, it is used as the primary seed. Take the primary seed and inoculate it onto nutrient broth medium, and culture it at 25-30℃ and 180-220 rpm for 12-14 hours. After passing the purity test, it is used as the secondary seed and stored at 2-8℃ for later use. The storage period is ≤5 days. A2. Preparation of bacterial culture: The culture was carried out in a fermenter. After sterilization, the Aeromonas hydrophila fermentation medium was inoculated with the secondary seed prepared in step A1 at 9-11% of the total volume of the medium. The culture was carried out at 25-30℃, with an aeration rate of 3.5-4.4 L / min and a stirring speed of 180-220 rpm for 10-12 hours to obtain the Aeromonas hydrophila seedling solution.

3. The method for preparing the grass carp Aeromonas hydrophila and Aeromonas vesiculosus bivalent inactivated vaccine for septicemia according to claim 2, characterized in that, The Aeromonas hydrophila fermentation medium contains the following components in the following amounts: bacteriological peptone 8-15 g / L, glucose 2-7 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate 0.2-0.6 g / L, beef extract 1-5 g / L, and sodium chloride 3-8 g / L; the solvent is water, and the pH is 7-8.

4. The method for preparing the grass carp Aeromonas hydrophila and Aeromonas verrucosa septicemia bivalent inactivated vaccine according to claim 1, characterized in that, The method for preparing the Aeromonas verrucosa seed culture includes the following steps: B1. Preparation of seeds for production: Take the frozen Aeromonas villi AVCI11 strain, inoculate it onto nutrient agar medium, and thaw and culture it at 25-30℃ for 18-24 hours; pick a single colony and streak it onto nutrient agar plates, and culture it at 25-30℃ for 18-24 hours. After passing the purity test, it is used as the primary seed. Take the primary seed and inoculate it onto nutrient broth medium, and culture it at 25-30℃ and 200 rpm for 12-14 hours. After passing the purity test, it is used as the secondary seed and stored at 2-8℃ for later use. The storage period is ≤5 days. B2. Preparation of bacterial culture: The culture was carried out in a fermenter. After sterilizing the Aeromonas versicolor fermentation medium, the secondary seed prepared in step B1 was inoculated at 9-11% of the total volume of the medium. The culture was carried out at 25-30℃, with an aeration rate of 3.5-4.4 L / min and a stirring speed of 180-220 rpm for 10-12 hours to obtain the Aeromonas versicolor seedling culture.

5. The method for preparing the grass carp Aeromonas hydrophila and Aeromonas vesiculosus septicemia bivalent inactivated vaccine according to claim 4, characterized in that, The Aeromonas verrucosa fermentation medium contains the following components in the following amounts: tryptone 8-12 g / L, glucose 4-6 g / L, potassium dihydrogen phosphate 1-4 g / L, magnesium sulfate 0.2-0.6 g / L, beef extract 2-5 g / L, and sodium chloride 3-7 g / L; the solvent is water, and the pH is 7-8.

6. The method for preparing the grass carp Aeromonas hydrophila and Aeromonas vesiculosus bivalent inactivated vaccine for septicemia according to claim 1, characterized in that, The specific steps for inactivation are as follows: add formaldehyde solution to the bacterial solution, inactivate at 35-38℃ for 30-35 hours, and stir at 40-60 rpm for 3-5 minutes every 3-6 hours during this period.

7. The method for preparing the grass carp Aeromonas hydrophila and Aeromonas vesiculosus bivalent inactivated vaccine for septicemia according to claim 6, characterized in that, The final concentration of formaldehyde in the bacterial solution is 0.2-0.3% vol.

8. The method for preparing the grass carp Aeromonas hydrophila and Aeromonas vesiculosus bivalent inactivated vaccine for septicemia according to claim 1, characterized in that, The adjuvant is aluminum hydroxide gel.

9. A grass carp aeromonas hydrophila and aeromonas versicolor septicemia bivalent inactivated vaccine prepared by the method according to any one of claims 1-8.

10. The grass carp Aeromonas hydrophila and Aeromonas vesiculosus bivalent inactivated vaccine for septicemia according to claim 9, characterized in that, The vaccine contains at least 2.0 × 10⁻⁶ live bacteria of each of Aeromonas hydrophila and Aeromonas vesiculosus before inactivation. 9 CFU / ml.

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