Introduction

Aeromonas infection, which affects a wide range of aquaculture animals, is a major limiting factor in fishery production. Aeromonas veronii and Aeromonas caviae are the main bacterial pathogens in cultured fish. They are widely present in aquatic and marine environments1 and can infect a large number of economically important fish, such as C. auratus and Oreochromis2, resulting in severe losses in the aquaculture industry. Vaccination is an effective and promising disease control strategy2. In the last decade, much research has been conducted on fish vaccines against Aeromonas veronii and Aeromonas caviae. For Aeromonas veronii, several subunit vaccines have been reported3, including inactivated vaccines. For example, A. veronii was inactivated by formaldehyde to prepare an inactivated A. veronii vaccine, and the immune effect of the inactivated A. veronii vaccine was comprehensively evaluated by acid phosphatase activity, superoxide dismutase activity, phagocytic activity, and other indicators4.

Most reported vaccines are monovalent and target only one pathogen. For aquaculture, vaccines to prevent and control many diseases would be ideal5. The trend of future vaccine development is to develop multivalent vaccines that are safe, efficient, and have a broad spectrum protective6,7. In perch, using an oral formulation of Vibrio bivalent revealed several mechanisms that enhanced protection for more than half a year when used with a new generation of adjuvants8.

Aeromonas disease are a serious threat to fisheries and cause considerable economic losses9. Aeromonas disease by A. veronii and A. caviae is one of the most common bacterial diseases in fisheries10. At present, A. veronii,and A. caviae and Aeromonas hydrophila for more than 85%, which causes extremely serious harm to fish species. In this study, A. veronii and A. caviae were selected for the preparation of a double vaccine, and their immune indices and immune protection rates were evaluated to provide a basic theoretical basis for the development of multiple vaccines.

Materials and methods

Animals and strains

C. auratus was purchased from a fish farm in Changchun (China). The average weight of the fish was 45.38 ± 1.35 g. After the fish were temporarily raised in the aquarium for two weeks, healthy C. auratus were selected for the experiment.

TH0426 strains of A. veronii, AC-CY strains of A. caviae, and Staphylococcus aureus (S. aureus) were preserved in Laboratory 211, College of Animal Science and Technology, Jilin Agricultural University. Th0426 Strains were collected at Pelteobagrus fulvidraco, GenBan entry number: GCA-001593245.1; (http:/www.ncbi.nlm.nih.gov/bioproject/293940). The AC-CY strain was collected from Hypophthalmichthys molitrix, and the genome sequence has been uploaded to NCBI under the accession number: CP139365-CP139367.

Ethical approval

All animal experiment procedures were strictly implemented in accordance with the “Regulations on the Administration of Laboratory Animals” approved by the State Council of the People’s Republic of China (1988.11.1) and the Jilin Provincial Animal Welfare and Research Ethics Committee. Jilin Agricultural University (JLAU08201409).This study is reported following ARRIVE guidelines.

Determination of the median lethal dose

Determination of the median lethal dose of A. Veronii and A. caviae

A. veronii and A. caviae cultured in LB liquid medium were centrifuged at 4000 r/min for 10 min, the supernatant was discarded, the mixture was poured into PBS, and the mixture was washed three times. Then, 100 µL of the original bacterial solution was added to 900 µL of PBS under aseptic conditions, and the above procedure was repeated after mixing uniformly. Several different concentrations were used for colony counting. The initial concentrations of the bacterial solutions were 1 × 108 CFU/mL, 1 × 107 CFU/mL, 1 × 106 CFU/mL, 1 × 105 CFU/mL, 1 × 104 CFU/mL and 1 × 103 CFU/mL.

Sixty C. auratus, 10 in each group, were divided into 6 groups. Six groups of C. auratus were injected intraperitoneally with the above six concentrations of bacteria, and the injection volume was 0.2 mL per fish. The animals were continuously observed for one week, and the number of daily deaths of C. auratus was recorded.

The semilethal concentrations of A. veronii and A. caviae were calculated by the improved Kirschner method. The formula is as follows:

$${\text{LogL}}{{\text{D}}_{{\text{5}}0}}={\text{Xk}} - {\text{i}}({\text{SP}} - 0.{\text{5}})$$
(1)

where Xk is the highest logarithmic dose, i is the difference between the logarithmic doses of the two adjacent groups, and P is the mortality of each dose group.

Optimal inactivation conditions for A. Veronii

Under aseptic conditions, a single colony of TH0426 cultured in RS media was selected and placed in 80 mL of liquid LB media. After the culture was completed, 50 µL of bacterial solution was absorbed by a liquid transfer gun and evenly spread on an agar-coated plate with RS media for colony counting under aseptic conditions. In 415 test tubes, 5 mL of bacterial suspension was added to each tube. Fifteen test tubes were equally divided into three groups, and 0.10%, 0.20%, 0.30%, 0.40%, or 0.50% formaldehyde was added to each group. The first group of bacterial solutions was stored at 4 °C for inactivation. The second group of bacterial solutions was inactivated in a 28 °C incubator, and the third group of bacterial solutions was placed in a 37 °C incubator for inactivation. At 6 h, 12 h, 18 h, 24 h, and 36 h after inactivation, the inactivated bacteria were cultured on an ultraclean worktable, and colony growth was observed and recorded11. The optimal inactivation conditions for A. veroniii were determined by evaluating the relationships between temperature, formaldehyde concentration, and inactivation time. The lowest formaldehyde concentration and shortest inactivation time was selected as the most effective conditions.

Study of the optimal inactivation conditions for A. Caviae

On an aseptic operating table, a single colony was selected from the RS identification medium and transferred to LB liquid medium. The samples were incubated at 30 °C for 18 h, after which colony counting was carried out. According to the colony count results, the final concentration of the bacterial solution was adjusted to 1 × 108 CFU/mL. The adjusted concentration of the bacterial solution was divided into 15 identical test tubes, and different concentrations of formaldehyde solution were added. The different concentrations of the preparation solution were put into a 4 °C refrigerator, 28 °C incubator, or 37 °C incubator for inactivation. At 6 h, 12 h, 18 h, 24 h, and 36 h after inactivation, 100 µL of the bacterial solution was evenly spread on LB solid culture media on a superclean worktable. The colonies were observed for colony growth on the media, and the results were recorded.

Preparation of combined vaccines containing A. Veronii and A. caviae

The inactivated A. veronii vaccine and A. caviae were centrifuged with sterilized PBS (pH = 7.2) 3 times to remove the formalin. PBS was added to adjust the bacterial concentrations to 2.0 × 109 CFU/mL, 4.0 × 108 CFU/mL, or 8.0 × 107 CFU/mL.

The same volume of A. veronii vaccine and A. caviae were mixed to make a combined vaccine. The final concentrations of the combined vaccine were 1.0 × 109 CFU/mL (high concentration), 2.0 × 108 CFU/mL (medium concentration), and 4.0 × 107 CFU/mL (low concentration) (the concentration of each of the two bacteria). The samples were stored in a refrigerator at 4 °C and set aside.

Sterility and safety test of the inactivated vaccine

Aseptic test: 100 µL of inactivated vaccine was extracted under aseptic conditions, smeared evenly on an agar-coated plate with RS culture media and LB solid media and incubated at 30 °C for 20 h to determine whether the bacteria grew on the plate.

Safety test: 20 healthy C. auratus were selected and divided into two groups. The first group received the inactivated vaccine, and the second group received sterile PBS. The method of intraperitoneal injection was adopted. The dose was 0.2 mL/tail. The morbidity and mortality of immunized C. auratus were recorded 7 days after inoculation.

Experimental design and grouping

  1. (1)

    Experimental grouping.


The experimental C. auratus were divided into four groups, A, B, C, and D, with 70 fish in each group. Group A was injected with the low-concentration vaccine, Group B with the medium-concentration vaccine, Group C with the high-concentration vaccine, and Group D with sterilized PBS.

  1. (2)

    Experimental design.


After the safety of the vaccine was determined, C. auratus was immunized by intraperitoneal injection, and the second booster immunization was carried out 14 days after the first immunization. The inoculation volume was 0.2 mL/tail. Samples were taken every 7 days before immunization and from postimmunization to prechallenge. During the sampling, 10 C. auratus were randomly selected for blood and liver, spleen, kidney, and intestinal tissue collection. Blood samples were collected and stored at 4 °C overnight, and the serum was collected by centrifugation for the detection of serum-related immune indices. The collected tissues were ground, and RNA was extracted and used for real-time fluorescence quantitative PCR detection.

Determination of leukocyte phagocytic activity

After blood collection, 0.2 mL of blood was taken from each group to prepare anticoagulant blood, and 30 µL of S. aureus was added. The blood was heated in a constant-temperature water bath, centrifuged, and the white cell layer was collected, stained, and observed under a microscope. The percentage of leukocyte phagocytosis (PP) was calculated by randomly observing 100 white cells. The calculation formula is as follows:

$${\text{PP}}=\left( {{\text{the }}\;{\text{Number}}\;{\text{ of}}\;{\text{ leukocytes}}/{\text{leukocytes}}\;{\text{ that}}\;{\text{ engulf}}\;{\text{ bacteria}}} \right) \times {\text{1}}00\%$$
(2)

Humoral immunity analysis

Enzyme-linked immunosorbent assay (ELISA) kits were used to measure the serum levels of IgM, SOD, CAT, AKP, LZM, ACP, fish complement 3 and fish complement 4 according to the manufacturer’s instructions.

Immune-related gene expression

A select number of C. auratus were randomly sampled from each group at three time points: prior to immunization (0 days), after immunization, and every 7 days before the challenge, and liver, spleen, kidney, and intestine tissues were collected after dissection. The collected fresh tissue was ground quickly, and RNA was extracted. After the extraction, the concentration of RNA was determined, and reverse transcription was carried out after normalizing the protein concentration in the reverse transcription mixture.

  1. (1)

    RNA reverse transcription.

The reverse transcription reaction system is shown in Table 1.

Table 1 The reverse transcription reaction system.
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  1. (2)

    qRT‒PCR detection.

The expression levels of four genes, IL-10, IL-1β, IFN-γ, and TNF-α, were determined. The internal reference gene used was β-actin. The specific primer sequences are shown in Table 2 below. Three replicates were established for each group, and the reference sample was 0 days.

Table 2 Primers sequences of real-time PCR.
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Determination of the relative immune protection rate

The challenge test was carried out on the 35th day, and the challenge concentration was 1 × 107 CFU/mL. The experimental groups were as follows:

Groups A, B, C, and D were divided into two groups, named A1, A2, B1, B2, C1, C2, D1, and D2 (20 fish in each group).

Group A1 was injected with 0.2 ml of A. veronii, and Group A2 was injected with 0.2 ml of A. caviae.

Group B1 was injected with 0.2 ml of A. veronii, and Group B2 was injected with 0.2 ml of A. caviae.

Group C1 was injected with 0.2 ml of A. veronii, and Group C2 was injected with 0.2 ml of A. caviae.

Group D1 was injected with 0.2 ml of A. veronii, and Group D2 was injected with 0.2 ml of A. caviae.

(2) After continuous observation for 14 days after the challenge, the number of dead C. auratus was recorded, and the relative percent survival (RPS) (was calculated as follows:

$${\text{RPS}}=\left( {{{1}} - {\text{Mi}}/{\text{Mc}}} \right) \times {{1}}00\%$$
(3)

where Mi is the mortality of the immune group and Mc is the mortality of the control group.

Statistical analysis

Statistical analysis was performed with SPSS v.16.0 and GraphPad Prism version 7.0 software. All data are presented as the mean ± SD (standard deviation) and were analyzed using one-way analysis of variance (ANOVA).

Results

Determination of the median lethal dose (LD50)

  1. (1)

    After different concentrations of A. veronii were injected into each group of C. auratus, the number of deaths within one week were recorded and are shown in Table 3. After different concentrations of Aeromonas veronii bacterial solution were injected into each group of C. auratuss, the mortality records within one week were as follows: All the C. auratuss died on the fourth day after being injected with a concentration of 1 × 108, with a mortality rate of 100%. All the C. auratuss died on the fifth day after being injected with a concentration of 1 × 107, with a mortality rate of 100%. Eight C. auratuss died on the fifth day after being injected with a concentration of 1 × 106, while two survived, with a mortality rate of 80%. Four C. auratuss died on the fourth day after being injected with a concentration of 1 × 105, while the others survived normally, with a mortality rate of 40%. None of the C. auratuss died after being injected with concentrations of 1 × 104 or 1 × 103. According to the improved Koch’s method, the half-lethal dose of A. veronii was 1.9 × 105 CFU/tail, which proved that the strain had strong pathogenicity.

Table 3 Determination of median lethal dose of crucian infected with A. veronii.
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  1. (2)

    The number of deaths one week after injecting different concentrations of A. caviae bacteria into each group of C. auratus is shown in Table 4. After different concentrations of Aeromonas veronii bacterial solution were injected into each group of C. auratuss, the mortality records within one week were as follows: All the C. auratuss died on the fourth day after being injected with a concentration of 1 × 108, with a mortality rate of 100%. All the C. auratuss died on the fifth day after being injected with a concentration of 1 × 107, with a mortality rate of 100%. Eight C. auratuss died on the fifth day after being injected with a concentration of 1 × 106, while two survived, with a mortality rate of 80%. Four C. auratuss died on the fourth day after being injected with a concentration of 1 × 105, while the others survived normally, with a mortality rate of 40%. None of the C. auratuss died after being injected with concentrations of 1 × 104 or 1 × 103. According to the improved Koch’s method, the half-lethal dose of A. caviae was 7.9 × 105 CFU/tail, which proved that the strain had strong pathogenicity.

Table 4 Determination of median lethal dose of crucian infected with A.caviae.
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Inactivation of A. Veronii

The inactivation times corresponding to different inactivation temperatures and formaldehyde concentrations are shown in Table 5. It can be seen from the table that when the inactivation temperature is 4 °C, the final concentration of formaldehyde is 0.2%, and it takes more than 36 h to achieve complete inactivation, while when the concentration of formaldehyde is 0.5%, it only takes more than 12 h to achieve complete inactivation. When the temperature was 28 °C and 37 °C, the final concentration of formaldehyde was 0.2%, and complete inactivation was achieved after 12 h. Finally, the selected inactivation temperature of A. veronii was 28 °C, the final concentration of formaldehyde was 0.2%, and the inactivation time was 12 h.

Table 5 A. Veronii inactivated time effect under different formalin and temperature.
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Inactivation of A. Caviae

The Table 6 illustrate the inactivation periods required at various temperatures and formaldehyde concentrations.It can be seen from the table that when the inactivation temperature is 4 °C, the final concentration of formaldehyde is 0.2%, and it takes more than 36 h to achieve complete inactivation, while when the concentration of formaldehyde is 0.5%, it only takes more than 12 h to achieve complete inactivation. At 28 °C and 37 °C, the final concentration of formaldehyde was 0.2%, and complete inactivation was achieved after 12 h. Finally, the selected inactivation temperature of A. caviae was 28 °C, the final concentration of formaldehyde was 0.2%, and the inactivation time was 12 h.

Table 6 A. Caviae inactivated time effect under different formalin and temperature.
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Sterility test and safety test

For the sterility test, 100 µL of inactivated vaccine was extracted under aseptic conditions and evenly spread on agar-coated plates with RS culture media and LB solid media. After 20 h of constant temperature culture at 28 °C, no colonies formed on the media.

Safety test: After vaccination and continuous observation for 14 days, the tested C. auratus grew well and exhibited no disease or death. No discernible differences were observed between the vaccinated group and the PBS control group. The experiment proved that the inactivated vaccine was nontoxic and not pathogenic to C. auratus.

Immune protection rate of vaccines

The challenge test was carried out on the 35th day, and the number of deaths in each group was recorded for 14 days, as shown in Table 7. The protection rate of the PBS group against A. veronii and A. veronii was 0%, the protection rate of the medium-concentration group was the highest, 75%, and the protection rate of the low-concentration group was the same as that of the high-concentration group (70%). The highest protection rate of A. caviae was 80% in the moderate-concentration group, and the lowest protection rate was 70% in the high-concentration group.

Table 7 The number of deaths in each group after poison attack.
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Leukocyte phagocytosis test

Three different regions were randomly selected under the microscope, the phagocytosis of 100 leukocytes was observed, the average value was obtained, and the percentage of leukocyte phagocytosis was calculated according to the formula. The results are shown in Fig. 1. The percentage of leukocyte phagocytosis in the PBS group did not significantly change during the whole immune cycle, but the PP value in the vaccine group increased significantly after the first immunization, especially in Group B, in which the PP increased from 12.77–30.09%0.10 The PP values peaked at 21 days, with Group B having the highest PP value (42.71%), Group C having the second highest(40.25%), and Group A having the lowest PP value (38.06%). PP began to decrease on the 28th day.

Fig. 1
figure 1

Percentage of leukocyte phagocytosis in PBS group, group A, group B, group C.

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Serum-specific antibody IgM detection

The level of IgM in the serum was determined by an ELISA kit at 0 days before immunization, every 7 days after immunization, and before the challenge. As shown in Fig. 2(a), the level of IgM antibody in C. auratus injected with the inactivated vaccine of A. veronii and A. caviae increased significantly and remained high after the second booster immunization, peaking at 21 days. The level of IgM antibody in Group A was the highest. During the whole immune period, there was no significant change in the level of IgM in the PBS control group, which was significantly different than what was observed in the vaccine group (P < 0.05).

Serum immune index test results

AKP concentration in serum

As shown in Fig. 2(b), the concentration of AKP in C. auratus in the three vaccine groups increased continuously after the first immunization, reached a maximum on the 21st day after the second immunization, decreased on the 28th day, and began to increase on the 35th day, among which the overall AKP level in the medium-concentration group was the highest. The level of AKP(P < 0.05)in the three vaccine groups was significantly greater than that in the PBS group. There was no significant change in AKP concentration in the PBS group.

C3 concentration in serum

The C3 concentration in the serum is shown in Fig. 2(c). After the first immunization, the C3 concentration of the three vaccine groups increased significantly, and the increase continued until before the second immunization. After the second immunization, the concentration of C3 in C. auratus serum began to increase slowly and reached a maximum on the 21st day, decreased on the 28th day, and increased on the 35th day in the low- and high-concentration groups. During the immune period, the concentration of C3 in the middle concentration group was slightly greater than that in the other two groups, and the C3 concentration in the A, B, and C vaccine groups (P < 0.05)was) was significantly greater than that in the PBS group. The concentration of C3 in the PBS group did not significantly change during the immune cycle.

C4 concentration in serum

The C4 concentration in the serum is shown in Fig. 2(d). After the first immunization, the C4 concentration in Groups A, B, and C increased continuously and reached a maximum on the 21st day; Group B had the highest concentration, Group C had the second highest concentration, and Group A had the lowest concentration. The concentration of C4 began to decrease on the 28th day and increased on the 35th day. During the immunization period, the concentration of C4 in the A, B, and C vaccine groups (P < 0.05) was significantly greater than that in the PBS group. There was no significant change in the concentration of C4 in the PBS group during the immunization period.

Serum CAT concentration

The concentration of CAT in the serum was detected, as shown in Fig. 2(e). After the first immunization, the concentration of CAT in Groups A, B, and C increased significantly, which lasted for 21 days and reached a maximum at 21 days. The concentration of CAT in Group B was slightly greater than that in the other two groups. The concentration of CAT began to decrease on the 28th day and increased slowly on the 35th day. During the immunization period, the concentration of CAT in Groups A, B, and C (P < 0.05) was significantly greater than that in the PBS group. There was no significant change in the concentration of CAT in the PBS group during the immunization period.

Serum SOD concentration

The concentration of SOD in the serum was detected, as shown in Fig. 2(f). After the first immunization, the concentration of SOD in Groups A, B, and C increased continuously, and the concentration of SOD in Groups B and C reached a maximum on the 21st day after the second immunization, while that in Group A reached a maximum on the 35th day. The concentration of SOD began to decrease on the 28th day and increased slowly on the 35th day. During the immunization period, the concentration of SOD in Groups A, B, and C (P < 0.05) was significantly greater than that in the PBS group. There was no significant change in the concentration of SOD in the PBS group during the immunization period.

Serum ACP concentration

The concentration of ACP in the serum was detected, as shown in Fig. 2(g). After the first immunization, the level of ACP in Groups A, B, and C increased significantly, and the level of ACP in the vaccine group increased continuously after the second immunization and reached a maximum on the 21st day. The level of ACP began to decrease on the 28th day and increased slowly on the 35th day. During the immunization period, the ACP levels in the A, B, and C (P < 0.05) vaccine groups were significantly greater than those in the PBS group. There was no significant change in the level of ACP in the PBS group during the immunization period.

LZM concentration in serum

The concentration of LZM in the serum was detected, as shown in Fig. 2(h). After the first immunization, the level of LZM in Groups A, B, and C increased significantly, the level of LZM in the vaccine group increased continuously after the second immunization, and the peak appeared on the 21st day. The LZM level began to decrease on the 28th day and increased on the 35th day. During the immunization period, the LZM levels in the A, B, and C (P < 0.05) vaccine groups were significantly greater than those in the PBS group. There was no significant change in the level of LZM in the PBS group during the immunization period.

Fig. 2
figure 2

Detection of serum related immune indicators in crucian carp. (a) IgM (b) AKP (c) C3 (d) C4 (e) CAT (f) SOD (g) ACP h: LZM.*p < 0.05,** p < 0.01.

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Real-time fluorescence quantitative PCR detection

In this study, the changes in the expression of IL-10, IL-1β, IFN-γ, and TNF-α in the tissues of immunized C. auratus were determined.

Relative IL-1β gene expression

Changes in IL-1β expression in the liver, spleen, kidney, and intestine were detected, as shown in Fig. 3. After the first immunization, the expression of IL-1β in all organs increased significantly. After the second booster immunization, the expression of IL-1β in all organ groups increased continuously and remained at a high level, peaking on the 21st day. During the whole immunization cycle, there was no significant change in the PBS group, but there was a significant difference between the inactivated vaccine group and the inactivated vaccine group (P < 0.01).

Fig. 3
figure 3

IL-1β relation expression of m RNA in different organisms, *p < 0.05,** p < 0.01, ***p < 0.001.

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Relative TNF-α gene expression

The expression of TNF-α in the liver, spleen, kidney, and intestine was detected, and the results are shown in Fig. 4. During the whole culture cycle, the expression of TNF-α increased significantly in the vaccine group but not in the PBS group, and there was a significant difference between the two groups (P < 0.01). After the first immunization, the expression of TNF-α in all organs increased significantly. With the progression of the second immunization, the expression of TNF-α in all tissues increased continuously and remained at a high level. The expression of TNF-α decreased on the 28th day. The expression of TNF-α in all tissues peaked on the 21st day and on the 21st, 28th, and 35th days. There was a significant difference in the expression of TNF-α among the tissues of the vaccine group. The expression of TNF-α in the liver and intestine of Group B was the highest (P < 0.05).

Fig. 4
figure 4

TNF-α relation expression of m RNA in different organisms, *p < 0.05,** p < 0.01, ***p < 0.001.

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Relative IFN-γ gene expression

The expression of IFN-γ in the liver, spleen, kidney, and intestine was detected, and the results are shown in Fig. 5. During the whole breeding cycle, the expression of IFN-γ in the vaccine group increased significantly, which was significantly different from that in the PBS group. After the injection of the inactivated vaccine, the expression of IFN-γ in all organs increased significantly. With the progression of the second immunization, the expression of IFN-γ increased continuously. The expression of IFN-γ decreased at 28 days and increased at 35 days. The expression of IFN-γ in each group peaked at 21 d. At 21 d, 28 d, and 35 d, there was a significant difference in the expression of IFN-γ among the vaccine groups. The expression of IFN-γ in Group B was the highest (P < 0.05).

Fig. 5
figure 5

IFN-γ relation expression of m RNA in different organisms, *p < 0.05,** p < 0.01, ***p < 0.001.

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Relative IL-10 gene expression

Liver, spleen, kidney, and intestinal tissues were collected to detect changes in IL-10 gene expression. Figure 6 shows that the expression of IL-10 in all organs increased significantly after the first immunization. With the progress of the second booster immunization, the expression of IL-10 in all tissues increased continuously and remained at a high level. The expression of IL-10 decreased on the 28th day and increased on the 35th day. The expression of IL-10 in all tissues peaked on the 21st day, and the expression in Group B peaked. There were significant differences in the expression of IL-10 in the liver among the vaccine groups at 21 d, 28 d, and 35 d. At the peak of the kidney and intestine, there was a significant difference in expression between Group A and Group B (P < 0.05). During the whole immunization cycle, there was no significant change in the expression of IL-10 in the PBS group, but there was a significant difference between the two groups (P < 0.01).

Fig. 6
figure 6

IL-10 relation expression of m RNA in different organisms, *p < 0.05,** p < 0.01, ***p < 0.001.

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Discussion

Inactivated vaccines are widely used in the aquaculture domain because of their simple production process, low cost, high safety, easy preservation, and easy transportation. Inactivated vaccines account for approximately 90% of global commercial fishery vaccines, and they are currently the most mainstream commercial aquatic vaccines. However, at present, most aquatic vaccines in China are univalent vaccines that can only treat diseases caused by a certain pathogen and cannot effectively prevent diseases caused by coinfection with multiple pathogens. The bivalent vaccine could enhance both the antibody titer level of A. veronii and A. caviaes as well as the survival rate of vaccinated fish. In addition, bivalent vaccines also reduce the timing of fish vaccination because they are able to protect against multiple bacteria through a single vaccination12. In this study, two pathogens, A. veronii and A. caviaes, were selected. After exploring the relationships between the inactivation time, temperature, and formaldehyde concentration, the optimum inactivation conditions were determined to be 28 °C. The final concentration of formalin was 0.2%, and the inactivation time was 12 h. Miyata13 reported that the best inactivation temperature for an A. hydrophila inactivated vaccine is 28 °C, and the inactivation effect of A. hydrophila is greatest at this temperature. This is consistent with the results of the inactivation temperature obtained in this study.

IgM is one of the most abundant immune proteins in teleost fish14. It mainly exists in the blood and strongly responds to pathogen infection or immune activation15. It can be used as a reference indicator of fish immunity. Immunoglobulin is produced by lymphocytes. When pathogenic bacteria invade the body, they can regulate the phagocytosis and immune response of humoral immunity so that fish can resist disease and defend against disease16. Its activity directly affects immune regulation and plays a vital role in the process of fish-specific immunity17. Klesius et al.18 used the immunoglobulin content to determine the immune function of fish for the first time. In this study, the IgM level in each vaccine group began to increase continuously after the first immunization and peaked after the second immunization, that is, at 21 days, while the IgM level in the PBS group did not significantly change. In a study by Vivas J et al.19, after rainbow trout were immunized with an inactivated A. hydrophila vaccine, the level of IgM increased significantly in a short time, which was significantly different from that of the control group. Vaccination can significantly improve the level of IgM and disease resistance in fish.

The metabolism of the fish is relatively constant, which forces the fish to produce a large amount of superoxide free radicals (O2−), causing harm to the fish. SOD in fish is an antioxidant enzyme that helps to scavenge free radicals. It is a natural scavenger of O2− in fish20, and its activity is also closely related to the immune ability of fish. Lysozyme (LZM) plays an important role in preventing disease, repairing wounds, and preventing infection in fish. Catalase (CAT) plays a scavenging role in the body, mainly by preventing poisoning caused by the excessive accumulation of hydrogen peroxide in fish. Acid phosphatase (ACP) mainly plays an important role in cell proliferation and differentiation and in detoxification systems. Alkaline phosphatase (AKP) is an important hydrolytic enzyme in fish21 that is directly involved in phosphorus metabolism. Its level is closely related to the growth and development of fish and the formation of ATP. C3 and C4 play important roles in the classical complement activation pathway. C3 can participate in the formation of a membrane-attacking complex (MAC) on the surface of target cells, which leads to lysis of the target cells. Analysis of the data in this study revealed that after the first immunization, the levels of CAT, SOD, LZM, ACP, AKP, C3, and C4 in C. auratus increased to varying degrees and were maintained at high levels after the second immunization. There was no significant change in the PBS group, and the immune index of each vaccine group was significantly greater than that of the PBS group. Chen S C et al.22. immunized Acipenser Schrenckii with an A. hydrophila inactivated vaccine and determined the activity of ACP and LZM in the serum. The results showed that after injection, the activity of the two enzymes increased to different degrees and was maintained at a high level, which was significantly different from that of the control group. Chang et al.23studied the immune effect of an Edwardsiella inactivated vaccine on yellow croaker. The results showed that the serum levels of CAT, SOD and complement C3 increased to varying degrees after immunization and were significantly greater than those in the control group. These findings are the same as the results of our study. According to our results, inactivated vaccine injection can enhance the immunity of fish and prevent them from being attacked by disease.

Changes in the expression of IL-1β and TNF-α can be used to evaluate the level of immune regulation, and they can interact with each other in the body, thus promoting the inflammatory response and immune response of the body24. The results showed that the expression of these two genes increased significantly after the first immunization and was maintained at a high level after the second booster immunization, and there was a significant difference between the two groups and the PBS group. This shows that inactivated vaccines can promote the expression of IL-1β and TNF-α. Ouyang et al.25 After tilapia were immunized with a heterologous Streptococcus agalactis extracellular product inactivated vaccine, the gene expression levels of the inflammatory cytokines IL-1β and TNF-α were determined. The results showed that the expression of the two genes increased to different degrees after immunization and challenge. This is consistent with the result we obtained.

The inflammatory cytokine IL-10 is produced by Th2 cells and plays an important role in immune regulation and defense against bacteria in the body26. The results showed that the expression of IL-10 in C. auratus in the experimental group increased significantly after the first immunization and maintained a high level after the second immunization, which was significantly different from that in the PBS group. This shows that inactivated vaccine injection can effectively increase the expression of the IL-10 gene in fish. IFN-γ, which is produced by Th1 cells, is the first line of defense used to induce an inflammatory response in the body and mainly plays antiviral and defensive roles27. The results showed that after the first immunization, the expression of IFN-γ in each organ tissue changed significantly and was significantly different from that in the PBS group. Within a week after the second booster immunization, the expression of IFN-γ peaked and was maintained at a high level, which indicated that the inactivated vaccine could effectively increase the expression of IFN-γ in C. auratus, similar to the findings of Yuan et al.28.

After the challenge, each vaccine group achieved suitable immune protection, among which the protection rate of the middle concentration group against A. veronii was the highest, reaching 75%, while that against A. caviae was 80%. The protection rate of the high-concentration group was the lowest, and the protection rate against A. veronii and A. caviae was 70%. The greater the concentration of the inactivated vaccine was, the greater the protective effect was. It is speculated that there may be a critical value. When the concentration of the inactivated vaccine exceeds this critical value, the protective effect will decrease, and the specific critical value needs to be further explored.

Many fish diseases caused by Aeromonas are caused by cross-infection of a variety of pathogens, but monovalent vaccines can only target diseases caused by a single pathogen, which is very restrictive29. The advantages of dual vaccines are evident. The immunization of fish can prevent and cure diseases caused by two kinds of pathogens at the same time. The combined vaccine prepared in this study can be used to control both A. veronii and A. caviae infection at the same time and has a strong protective effect. However, compared with other monovalent vaccines, the immune protection rate is still too low. For example, Klesius et al.30immunized koi with an inactivated vaccine of A. veronii, and the protection rate reached 93.7%. Doan et al.31 systematically studied the safety, immune protection, and immune titer of a carp vertical scale disease vaccine. The results showed that the immune protection rate of the injection route was 88.9%. This is much higher than the protection rate of the vaccine we developed. It is speculated that the dual vaccine may have produced some reactions in C. auratus, resulting in a decrease in the immune effect. In actual production, we can try to improve the immune protection rate of the combined vaccine by adding adjuvants.

Conclusion

A combined A. veronii and A. caviae inactivated vaccine was successfully prepared. The optimal inactivation conditions for the vaccine were 28 °C, 0.2% formalin concentration, and 12 h inactivation time. Immune index determination and challenge tests confirmed that the immune protection effect of the medium-concentration vaccine group was the greatest; the protection rate against A. veronii was 75%, and the protection rate against A. caviae was 80%. This study provides a theoretical basis for the development of vaccines for aquaculture.