The experimental Carassius auratus were purchased from a fish farm in Changchun, with each fish weighing an average of 50 ± 5 g. After disinfection of the fish surfaces through immersion in potassium permanganate and saline solution, they were temporarily housed in aquarium tanks in the Aquaculture Laboratory of Jilin Agricultural University. Consistent oxygen supply and temperature were maintained in the tanks, with one-third of the water being changed every 2 ds. Once the fish had stable growth and adapted to the water environment, they were randomly assigned to groups for bacterial infection and immunity experiments.

In earlier studies, a live attenuated vaccine strain, designated as ΔhisJ, was developed by knocking out the hisJ gene in the wild type A.veronii TH0426 (GenBank accession no. CP012504.1) (21).

The wild-type A.veronii TH0426 (which are Ampicillin-resistant) and the attenuated vaccine strain ΔhisJ, preserved in the laboratory, were streaked onto RS agar plates (Hopebiol, Qingdao, China) and incubated at 30°C for 12 h. Single colonies were selected and inoculated into liquid LB medium (containing 0.1% Ampicillin), then cultured at 30°C with agitation at 180 rpm for 12 h. The bacterial culture was subsequently centrifuged at 6000 rpm for 5 min; the supernatant was discarded, and the pellet was washed three times with PBS and resuspended in the same volume. Finally, 25% glycerol was added, and the mixture was thoroughly mixed before being stored at −80°C in an ultra-low-temperature refrigerator. The next day, a portion of the frozen bacterial culture was retrieved and counted using a serial dilution technique. The process involves diluting a bacteria sample several times to break the sample into manageable concentration levels. After each dilution, a portion of the sample may be plated onto a LB agar plate, and after incubation, the colonies are counted. This step was crucial as it preceded the quantitative determination of the average colony-forming units per milliliter (CFU/mL), a critical measure determining the bacterial concentration used for subsequent challenge and vaccinations.

Employing the method outlined above, Staphylococcus aureus CMCC(B)26,003 was cultivated and enumerated, with the culture conditions maintained at a temperature of 37°C. The bacterial culture was then adjusted to a concentration of 1 × 10⁷ CFU/mL, suitable for subsequent phagocytosis assays.

The vaccine strain ΔhisJ is being tested for genetic stability over multiple generations of growth in LB medium, using PCR amplification. The strain ΔhisJ was continuously passaged for 45 generations in LB liquid medium containing 0.1% Ampicillin, with genome extraction every 5 generations. At the same time, PCR identification was performed using the primers hisJ-for/hisJ-rev (Table 1). The target bands were sent to Kumei (Changchun) Biotech Co., Ltd. for sequencing. The purpose of the experiment is to confirm that the mutant remains consistent over many generations, ensuring the strain’s stability for any application in vaccine development.

To determine the optimal immune dosage for immunizing Carassius auratus and confronting them with a bacterial challenge, we employed various administration routes for the wild-type strain A. veronii TH0426 and the live attenuated vaccine ΔhisJ, including intraperitoneal and muscular injections, along with immersion and oral routes. A total of 120 Carassius auratus, evenly divided into 8 groups, were immunized with different types and doses of vaccines. Over a period of 14 ds, meticulous daily monitoring was conducted to record the mortality rates of Carassius auratus. Subsequently, these data were utilized to calculate the LD₅₀ for the respective bacterial strains employing the modified Karber method. The calculation formula is as follows:

LD₅₀ = log-1[Xm − i(p − 0.5)], note: i is the dose interval (i.e., the difference between the logarithmic doses of two adjacent dose groups); Xm is the logarithm of the maximum dose; p is the mortality rate of each dose group (mortality rates are all expressed in decimals); p is the sum of the mortality rates of each dose group.

Different types of vaccines are prepared according to the methods described below. Both strains, TH0426 and ΔhisJ, were initially cultured in LB medium before being washed and resuspended in PBS. TH0426 was administered through intraperitoneal injection, each injection consisting of 200 μL of bacterial solution at concentrations ranging from 2 × 10⁸ to 2 × 10⁴ CFU/mL. In contrast, ΔhisJ was injected either intraperitoneally or into the muscle near the dorsal fin, with each injection being 200 μL and concentrations varying from 2 × 10⁹ to 2 × 10⁵ CFU/mL. During the immersion tests, the TH0426 and ΔhisJ culture was added to the aquarium, with the fish being immersed for 30 min in solutions containing bacterial concentrations ranging from 2 × 10⁸ to 2 × 10⁴ CFU/mL, and from 2 × 10⁹ to 2 × 10⁵ CFU/mL. Following this treatment, the fish were relocated to a bacteria-free aquarium for subsequent rearing. For the oral tests, the concentration of ΔhisJ was adjusted to levels ranging from 2 × 10⁸ to 2 × 10⁴ CFU/mL, and from 2 × 10⁹ to 2 × 10⁵ CFU/mL, respectively. Every 200 μL of this bacterial solution was combined with 100 g of fish feed (supplied by TONGWEI Co., LTD., China). Additionally, to ensure the bacteria were adequately encapsulated, 3 g of sodium alginate powder (sourced from Sigma-Aldrich, United States) was used at a 1.5% ratio during the preparation of the fish feed. After the encapsulation process, which involved drying, the feed was refrigerated at 4°C and preserved for future feeding sessions.

For vaccines administered by intraperitoneal (IpV group) or intramuscular injection (ImV group), the concentration of the attenuated vaccine strain ΔhisJ is 2 × 10⁷ CFU/mL. For vaccines used in immersion immunization (IV group), the culture of ΔhisJ is added to the aquarium to achieve a concentration of 2 × 10⁷ CFU/mL. For vaccines used in oral immunization (OV group), the concentration of the ΔhisJ is adjusted to 2 × 10⁷ CFU/mL, and for every 200 μL of bacterial solution, it is mixed with 100 g of fish feed.

To assess the safety of the attenuated vaccine strain ΔhisJ, the Carassius auratus were randomly divided into five groups, with 10 fish in each group, for a challenge test. For the IpV group and the ImV group, the dose of the vaccine was 200 μL per fish. For the IV group, the Carassius auratus were first soaked in an aquarium containing the attenuated vaccine for 30 min, and after the immunization, the fish were transferred to a vaccine-free aquarium. For the OV group, fish were fed vaccine-coated feed at a dose of 2% of body weight, continuously for 5 ds, and after the immunization, they were fed non-vaccine-coated feed. After the immunization, continuous observation was carried out for 1 week, and the clinical symptoms and mortality rate of Carassius auratus in each group were recorded. Carassius auratus were intraperitoneally and intramuscular injected with an equal volume of PBS as a control group (namely Control group).

Carassius auratus were divided into 7 groups, each consisting of 60 fish. In the intraperitoneal vaccine group (IpV group) and the intramuscular vaccine group (ImV group), the fish were subjected to either intraperitoneal injections or dorsal fin muscle injections of the attenuated vaccine strain ΔhisJ. A second dose of the vaccine was administered on the 14th d, following the same procedures. For the immersion vaccination group (IV group), the fish were immersed in an aquarium with a 2 × 10⁷ CFU/mL concentration of the vaccine. After soaking for 30 min, they were transferred to a vaccine-free aquarium. A second round of immersion vaccination was conducted 14 ds later. In the oral vaccine group (OV group), each fish received vaccine-coated feed amounting to 2% of its body weight. This method of oral immunization continued for 5 ds, followed by another 5 ds session starting on the 14th day. After immunization, the fish were given feed not coated with the vaccine.

For both the intraperitoneal PBS group (IpP group) and the intramuscular PBS group (ImP group), the Carassius auratus were injected with 0.2 mL of PBS per fish, either intraperitoneally or in the dorsal fin muscle, with a follow-up injection on the 14th d. Furthermore, a Blank Control group (BC group) was established in which the fish did not receive any form of vaccine immunization or PBS injections.

For serum samples, blood was drawn from the caudal vein of 10 randomly chosen healthy Carassius auratus on the 0, 7th, 14th, 21st and 28th d post immunization. The collection was performed post-anesthesia. Half of the obtained blood was treated with a sodium heparin solution (heparin sodium 1,000 IU/mg in 0.85% NaCl) to serve as an anticoagulant, intended for subsequent leukocyte phagocytic activity analysis. The remaining blood was subjected to centrifugation at 3000 rpm for 15 min to separate the supernatant, earmarked for evaluating various serum immune indices and antibody levels.

As for tissue samples, after the blood collection, the same fish were dissected to harvest specific organs and tissues, including the liver, kidneys, spleen, intestines, and gills. These specimens were promptly stored in a −80°C freezer, preserving them for future RNA extraction processes and cytokine assays.

According to the manufacturer’s instructions, we utilized ELISA kits from the Nanjing Jiancheng Bioengineering Institute, Jiangsu, China, to analyze the enzyme activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM). One Ginoff unit of AKP and ACP activity corresponds to 1 mg of phenol produced in 100 mL of serum reacting with the substrate at 37°C for 30 min, with the absorbance measured at 520 nm. One unit of SOD activity refers to the amount of enzyme that results in a 50% inhibition rate of SOD in the reaction system, measured by the absorbance at 450 nm. One unit of LZM activity was defined as the amount of lysozyme that causes a 0.001 unit reduction in absorbance per minute at 530 nm. All analyses were conducted in triplicate.

Based on the instruction manuals of the fish IgM ELISA kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), and the fish complement proteins C3 and C4 ELISA kits (Yan Domain Chemical Reagent Co., Ltd., Shanghai, China), the levels of IgM antibodies, C3 and C4 in serum were measured using a double-antibody sandwich ELISA method. The coating micropores provided in these kits were pre-coated with mouse anti-carp IgM, complement C3, and complement C4 antibodies. Standards, serum samples, and horseradish peroxidase (HRP)-labeled detection antibodies were added sequentially to the wells of the ELISA plate, followed by incubation and washing. Subsequently, tetramethylbenzidine (TMB) substrate solution was added for color development, and the reaction was stopped with H₂SO₄. The optical density (OD) was measured using a spectrophotometer at a wavelength of 450 nm. This value positively correlated with the corresponding antibodies in the sample. The concentration of complement C3 and C4 in the samples was calculated using a standard curve. All analyses were conducted in triplicate.

Using a 96-well V-bottom plate to determine the agglutination titer of serum, the level of specific antibodies is evaluated. The specific procedure is as follows:

A.veronii TH0426 is cultured until it reaches the logarithmic phase. After centrifugation, it is washed three times with PBS, and the bacterial suspension’s concentration is adjusted to 1 × 10⁷ CFU/mL. The bacteria are then inactivated by heating at 56°C for 30 min, serving as the inactivated bacterial antigen for the test. Take a 96-well plate and add 80 μL of saline solution to the first well, and 50 μL of saline solution to the remaining wells. Then add 20 μL of serum to the first well. Mix it evenly, then transfer 50 μL from this well to the second well. Using the same method, continue the process until the penultimate seventh well, discarding 50 μL each time. This creates a series of dilutions of the serum antibodies in the wells at 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512 concentrations. The eighth well functions as a control and does not contain any serum. Distribute 50 μL of the deactivated bacterial antigen into every well, ensuring thorough mixing. Subsequently, allow the plate to incubate at 37°C for a duration of 2 h, and then let it remain at ambient temperature throughout the night. In the final step, assess the extent of bacterial agglutination through microscopic examination.

Staphylococcus aureus CMCC(B)26,003 was inoculated into TSB medium (Hopebio, Qingdao, Shandong, China) and subsequently washed three times with PBS. The bacterial suspension was then adjusted to a concentration of 1 × 10⁷ CFU/mL, used for phagocytic antigen. We then mixed 0.2 mL of anticoagulated blood with 0.02 mL of the prepared phagocytic antigen. This mixture was incubated in a water bath set at 25°C for 60 min, with gentle agitation every 10 min to ensure uniform consistency. After incubation, the mixture was centrifuged at 4000 rpm for 10 min, resulting in the formation of distinct layers. We carefully pipetted the intermediate layer enriched with white blood cells, depositing 5–10 μL droplets onto a glass slide to create a blood smear. The smear was allowed to air-dry and was then secured for the Wright-Giemsa staining process. We applied the stain to the blood smear for 10 min. Any residual stain was removed with distilled water, and the slide was left to dry completely. For the analysis, the slide was positioned under a microscope. We chose a random field of vision to scrutinize the white blood cells, evaluating their phagocytic activity towards bacteria. This observation was pivotal for determining the Phagocytic Percentage (PP) of white blood cells using the formula Equation 1:

RNA was extracted from the liver, kidneys, spleen, intestines, and gills of Carassius auratus following vaccination, according to the instructions of the RNA extraction kit (TaKaRa, Beijing, China). Reverse transcription was carried out using a reverse transcription kit (TaKaRa, Beijing, China) to obtain cDNA. The cDNA obtained from reverse transcription served as a template for qPCR, performed with the TB Green^® Premix Ex Taq^™ II kit (TaKaRa, Beijing, China). The primer sequences for each cytokine are presented in Table 1. RNA-free water was added for the negative control, and β-actin was used as the internal reference gene. The relative expression levels of each gene were calculated using the 2^−ΔΔCT method. All qRT-PCR reactions were set up in triplicate.

On the 35th d after the first immunization, 25 fish from each group were randomly selected for the challenge test by intraperitoneal injection of A.veronii TH0426. The immune protective effect in each experimental group was observed. A.veronii TH0426 was revived and, after bacterial counting, the concentration was adjusted to 1.2 × 10⁷ CFU/mL to serve as the challenge bacterial solution. Each Carassius auratus was injected intraperitoneally with a dose of 0.2 mL. The experiment and control groups were observed for 14 ds, and any dead fish were promptly removed and recorded.

The relative percent survival for each group was calculated based on the mortality rates using the formula:

On the 1st, 3rd, and 5th d following the challenge, liver, spleen, and kidney tissues were collected from moribund fish in each group as samples for testing. The organs were weighed, and then 0.4 g of tissue from each organ was randomly cut and placed into an EP tube containing 4 mL of sterile PBS. After mincing, the tissues were homogenized, and the homogenate was diluted in a 10-fold gradient and then spread onto RS agar plates. Following overnight incubation at 37°C, colony counting was conducted. The bacterial load in the liver, spleen, and kidneys at different time intervals for each experimental group was calculated to assess the immune protection efficacy of the vaccine.

Statistical analyses were conducted using SPSS 26.0 and GraphPad Prism v9.5.0, employing one-way Analysis of Variance (ANOVA) followed by t-tests. Data are presented as mean ± standard error. Significant differences are indicated with an asterisk (*), where *p < 0.05, **p < 0.01, and ***p < 0.001 are considered to represent statistically significant differences in mean values.

To assess the genetic stability of the hisJ-deleted strain, the ΔhisJ was serially passaged for 45 generations, and PCR amplification was conducted using the primers hisJ-for and hisJ-rev. In the wild type strain A.veronii TH0426, a 2,648 bp target band (including the ORF of hisJ) was amplified. In the ΔhisJ across different generations, a consistent 1874 bp target band was amplified, as shown in Figure 1. This result aligns with the expectations, demonstrating that the hisJ-deleted strain has good genetic stability.

To determine the immunization and the challenge dose, the LD₅₀ of A.veronii TH0426 and ΔhisJ. The results (Table 2) showed that under different administration routes, the LD₅₀ of ΔhisJ significantly increased compared to the wild type strain TH0426. Specifically, in the IpV group, the LD₅₀ increased by 357.8 times (2.10 × 10⁸/5.87 × 10⁵), the largest increase, while in the IV group, it increased by 146.9 times (2.16 × 10⁸/1.47 × 10⁵), the smallest increase. Across the four inoculation routes, the highest bacterial concentration at which Carassius auratus did not die when inoculated with ΔhisJ was 2 × 10⁷ CFU/mL. The maximum LD₅₀ for the wild-type strain TH0426 was 3.55 × 10⁶ CFU/tail. It is noteworthy that under the IpV injection route, the LD₅₀ for fish was the smallest, indicating that intraperitoneal injection is the most virulent route for the strain in fish. Therefore, this study chose a dose of 4 LD₅₀ (≈2.4 × 10⁶ CFU/tail) for intraperitoneal injection to conduct immune protection experiments. This involved adjusting the concentration of ΔhisJ to 2 × 10⁷ CFU/mL with an immunization dose of 0.2 mL, and adjusting the concentration of TH0426 to 1.2 × 10⁷ CFU/mL with a virulent dose of 0.2 mL. The clinical symptoms exhibited by Carassius auratus infected with the wild-type strain A.veronii TH0426 and the vaccine ΔhisJ included darkening of the body, slowed swimming, floating to the surface, surface bleeding, abdominal swelling, and in severe cases, skin ulcers.

In the groups of Carassius auratus immunized via intraperitoneal vaccination (IpV), intramuscular vaccination (ImV), immersion vaccination (IV), oral vaccination (OV), and intraperitoneal PBS injection (IpP), there were no instances of death following the administration of the vaccine or PBS. Fish in the IpV and ImV, groups exhibited strong stress responses, showing signs of poor mental state, reduced feeding, slow swimming, and floating to the water surface, but they returned to normal the next day. Fish in the IV group showed mild stress responses, with a slight reduction in feeding, but recovered to normal 6 h after immunization. Fish in the OV group did not show any stress responses. Upon dissection of fish from each group, there were no obvious pathological changes observed in the intestines, gills, kidneys, spleen, or liver of any group (Figure 2A). Notably, some fish in the ImV group experienced partial scale loss and even swelling and ulceration at the injection site (Figure 2B).

After immunizing Carassius auratus with the vaccine strain ΔhisJ, non-specific immune related molecules in the serum of the Carassius auratus were detected, including IgM, ACP, AKP, LZM; complement C3, complement C4, and SOD. Taking IgM as an example, the results (Figure 3A) show that the IgM content in the serum of Carassius auratus in various vaccine-immunized groups generally showed an upward trend after the primary immunization, and continued to increase after the secondary immunization. The IgM content in the serum of Carassius auratus in the IpV group, OV group, and IV group showed a continuous upward trend from the start of the initial immunization, reaching their respective peaks on the 28th d, then the IgM content began to decline, and by the 35th day after immunization, compared to the control group, the IgM content in the serum of the Carassius auratus in each immunized group was still significantly increased (p < 0.001). The IgM content in the ImV group showed a continuous upward trend from the 0 day of the primary immunization until the 21st d, reaching a peak, and then showed a downward trend. By the 35th d post immunization, compared to the control group, the IgM content in the immunized groups of Carassius auratus significantly increased (p < 0.001). It is worth noting that, on the 28th and 35th d post immunization, the non-specific immune molecules in the IpV group were higher compared to the other three vaccine immunization groups, suggesting that the intraperitoneal injection immunization route stimulates Carassius auratus to produce a more powerful non-specific immune effect. The BC group, IpP group, and ImP group showed no obvious trends in IgM content during each immunization period (p > 0.0.05), and there was little difference in IgM content in the serum between the groups. The trends in the levels of ACP, AKP, LZM, complement C3, complement C4, and SOD in the serum were the same as those of IgM.

Using the 96-well hemagglutination assay, the specific antibody titers of Carassius auratus serum immunized via different routes were tested. The results showed (Figure 4) that there were no significant differences in serum titers among the control groups (BC group, ImP group, and IpP group). The specific antibody titers of the Carassius auratus in each group immunized via different routes (IpV group, ImV group, IV and OV groups) were all higher than those of the control groups. Interestingly, the serum titer gradually increased with the increase of immunization times and the extension of time. On the 28th d, the serum antibody titer of the vaccinated Carassius auratus reached its peak and then declined. Among these, the serum antibody titers of the Carassius auratus in the IpV group were always the highest on the 7th, 14th, 21th, and 28th d post immunization. The antibody titers in both the OV and ImV groups rank second in terms of magnitude, with the group receiving immersion immunization displaying the lowest antibody titers. The results indicated that all four vaccine immunization routes could stimulate Carassius auratus to produce specific humoral immunity, but the intraperitoneal injection route of vaccination had the best immunization effect.

The anticoagulated blood of Carassius auratus post-vaccine immunization was taken and Staphylococcus aureus CMCC(B)26,003 was added for a white blood cell phagocytosis experiment. The phagocytosis of 100 randomly observed white blood cells under a microscope was counted. The image of white blood cells engulfing bacteria is shown in Figure 5A, and the phagocytic ratio is displayed in Figure 5B. On the 0 d, the phagocytic ratio ranged between 10.01–13.73%, with no significant differences among the seven groups including the control group. However, following the vaccination of the vaccine test groups, from the 7th d of immunization, there was a significant upward trend in the phagocytic ratio of each vaccine immunization group, followed by a slight decrease on the 14th d. With the secondary immunization, the white blood cell phagocytic ratio continued to rise, reaching the peak phagocytic percentage for each vaccine test group on the 21st or 28th d of immunization, followed by a declining trend. On the 21st d post immunization, the white blood cell phagocytic percentage of the muscle ImV group increased significantly compared to the ImP group (p < 0.001); on the 28th d, the white blood cell phagocytic percentage of the IpV group increased significantly compared to the IpP group (p < 0.001); on the 28th d, the white blood cell phagocytic percentage of the oral vaccine group (OV) significantly increased compared to the BC group (p < 0.001); the white blood cell phagocytic percentage of the IV group showed an upward trend, but only on the 21st and 28th d post immunization, the phagocytic percentage was significantly different from the BC group (p < 0.05), with no significant differences at other time points. The white blood cell phagocytic ratios of the ImV and IpV groups were higher than those of the IV and OV groups, and the IpV group was higher than the ImV group. These results indicate that the intraperitoneal injection immunization route induced a more intense immune response.

Before immunization (on the 0 d), and on the 7th, 14th, 21st, 28th, and 35th d after the first vaccine immunization, Carassius auratus were randomly selected from each group for dissection. The liver, spleen, kidney, intestine, and gills were harvested to extract RNA, and qRT-PCR was performed to analyze the relative expression levels of cytokines IL-1β, TNF-α, IL-10, and TGF-β. The expression levels of the four cytokines in the liver were tested and analyzed. The results showed that (Figure 6), at all the testing days except the 0 d, the expression levels of IL-1β and TNF-α in the OV and IV groups were higher than those in the BC group. The highest levels in the OV, IV, and IpV groups were reached on the 28th d post-immunization, while for the ImV group, the peak was on the 21st d, followed by a decline in expression levels. The expression levels of cytokines in the spleen, kidney, intestine, and gills showed the same trends as those in the liver. Interestingly, the highest expression levels of the four cytokines in different organs of fish in the IpV group (on the 28th d) were higher than those in the other three vaccine immunization groups. The highest expression levels of the four cytokines in the ImV group fish (on the 21st d post-immunization) were higher than those in the OV and IV groups but still lower than those in the IpV group on the 28th d.

The Carassius auratus in each group were intraperitoneally injected with the wild strain A.veronii TH0426 on the 35th d after immunization to measure the immune protection effect. The Carassius auratus in the IpP, ImP and BC group all died within 6–7 ds after the challenge, exhibiting typical characteristics such as scale loss, bleeding, and abdominal swelling. Some fish in the various vaccine immunization groups also died within 6–7 ds after the challenge. As shown in Figure 7, On the 14th d after the challenge, the survival rate of the fish was 64% in the IpV group, 56% in the ImV group, 52% in the OV group, and 48% in the IV group. The results indicate that Carassius auratus immunized with the vaccine strain ΔhisJ have a certain protective effect against A.veronii infection. The protection rates vary with different immunization routes. The most effective immunization route is intraperitoneal injection, followed by muscle injection, oral immunization, and soaking immunization, in decreasing order of protection effectiveness.

After intraperitoneal injection with the wild strain A.veronii TH0426 in each immunized and control group of Carassius auratus, the liver, spleen, and kidney of dying fish were collected on the 1st, 3th, and 5th d post-challenge to measure the bacterial load in these organs. The results showed that (Figure 8) on the 1st day post-challenge, there was no significant difference in bacterial load compared to the respective control groups. However, on the 3th and 5th d post-challenge, the bacterial load in the organs of fish in the immunized groups was generally lower than that in their respective control groups. Additionally, the bacterial load on the 5th d was lower than on the 3th d, indicating a decreasing trend in bacterial load over time in the immunized groups. In contrast, the bacterial load in the liver, spleen, and kidney of the control group fish showed a trend of increase or maintained a high level without decreasing. Since the control group fish, lacking protective immunity, all died within 7 ds post-challenge, measuring bacterial load in their organ tissues on the 7th d was not feasible; hence, further analysis of the load was not conducted. The results of the bacterial load tests indicate that the vaccine strain ΔhisJ could gradually restrict the rapid proliferation of bacteria in the organ tissues of Carassius auratus, reducing the bacterial load in these tissues, thereby providing a certain level of immune protection against A.veronii. Notably, there were significant differences in the bacterial load in the organs of Carassius auratus immunized via different routes with the vaccine strain ΔhisJ. Fish vaccinated via intraperitoneal injection had the lowest bacterial load in their organs on the 3th and 5th d post-challenge, followed by those in the ImV group, the OV group, and the IV group.