Immunogenicity and protective ef ﬁ cacy of OmpA subunit vaccine against Aeromonas hydrophila infection in Megalobrama amblycephala : An effective alternative to the inactivated vaccine

Aeromonas hydrophila is a kind of zoonotic pathogen, which can cause bacterial septicemia in ﬁ sh and bring huge economic losses to global aquaculture. Outer membrane proteins (Omps) are conserved antigens of Aeromonas hydrophila , which can be developed as subunit vaccines. To evaluate the protective ef ﬁ cacy of inactivated vaccine and recombinant outer membrane protein A (OmpA) subunit vaccine against A. hydrophila in juvenile Megalobrama amblycephala , the present study investigated the immunogenicity and protective effects of both vaccines, as well as the non-speci ﬁ c and speci ﬁ c immune response of M. amblycephala . Compared with the non-vaccinated group, both inactivated and OmpA subunit vaccines improved the survival rate of M. amblycephala upon infection. The protective effects of OmpA vaccine groups were better than that of the inactivated vaccine groups, which should be attributed to the reduced bacterial load and enhanced host immunity in the vaccinated ﬁ sh. ELISA assay showed that the titer of serum immunoglobulin M (IgM) speci ﬁ c to A. hydrophila up-regulated signi

Aeromonas hydrophila is a kind of zoonotic pathogen, which can cause bacterial septicemia in fish and bring huge economic losses to global aquaculture. Outer membrane proteins (Omps) are conserved antigens of Aeromonas hydrophila, which can be developed as subunit vaccines. To evaluate the protective efficacy of inactivated vaccine and recombinant outer membrane protein A (OmpA) subunit vaccine against A. hydrophila in juvenile Megalobrama amblycephala, the present study investigated the immunogenicity and protective effects of both vaccines, as well as the non-specific and specific immune response of M. amblycephala. Compared with the non-vaccinated group, both inactivated and OmpA subunit vaccines improved the survival rate of M. amblycephala upon infection. The protective effects of OmpA vaccine groups were better than that of the inactivated vaccine groups, which should be attributed to the reduced bacterial load and enhanced host immunity in the vaccinated fish. ELISA assay showed that the titer of serum immunoglobulin M (IgM) specific to A. hydrophila up-regulated significantly in the OmpA subunit vaccine groups at 14 d post infection (dpi), which should contribute to better immune protective effects. In addition, vaccination enhanced host bactericidal abilities might also attribute to the regulation of the activities of hepatic and serum antimicrobial enzymes. Moreover, the expression of immune-related genes (SAA, iNOS, IL-1 b, IL-6, IL-10, TNF a, C3, MHC I, MHC II, CD4, CD8, TCR a, IgM, IgD and IgZ) increased in all groups post infection, which was more significant in the vaccinated groups. Furthermore, the number of immunopositive cells exhibiting different epitopes (CD8, IgM, IgD and IgZ) that were detected by immunohistochemical assay had

Introduction
Aeromonas hydrophila is a kind of gram-negative bacteria with strong pathogenicity, which can infect human, livestock and aquatic animals. It is easy to proliferate and cause disease outbreaks in the aquaculture ponds during high temperature seasons, causing huge economic losses to the global aquaculture industry (1). A. hydrophila exhibits a wide range of pathogenicity, especially the explosive septicemia that has been found in most freshwater fish, such as Megalobrama amblycephala, Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cyprinus carpio and the like, which exhibits the typical pathological features of surface hyperemia, anal redness and swelling with hemolysis (2). Its pathogenic factors mainly include exotoxins, extracellular enzymes, outer membrane proteins (Omps), S-layer proteins, and lipopolysaccharides (3). A. hydrophila commonly infects animals via adhesion and enterotoxic mechanisms, proliferating in the intestines and migrating to other tissues through blood circulation, and then synthesizing and secreting exotoxins, causing tissue lesions, systemic symptoms and death. At present, antibiotics are usually used to prevent and treat bacterial septicemia, but long-term use of antibiotics will not only cause antimicrobial resistance, but also affect the safety of aquatic products. Vaccine immunization is an important and environmentally effective way for disease prevention and control in aquaculture, and the significance of vaccine development and application is more prominent under the background of antibiotics reduction and substitution. So far, many studies focus on A. hydrophila vaccines, including whole bacteria inactivated vaccine (4), subunit vaccine (5), attenuated live vaccine (6), nucleic acid vaccine (7), etc., and has gone through the developmental stages from univalent vaccine to multivalent vaccine (8,9).
Previous studies have found that the inactivated A. hydrophila vaccines provide relatively excellent immune protective effects on aquatic animals by inducing host immune responses. For instance, the inactivated A. hydrophila vaccine shows a significant immune protective effect on juvenile Ictalurus punctatus, with the specific antibody that has been induced and produced post immunization (4). Similarly, vaccination with inactivated A. hydrophila vaccine by intraperitoneal injection or immersion shows significant upregulation of the skin mucus lysozyme and specific antibody levels in the serum and skin of Piaractus mesopotamicus, thereby increasing the survival rate post infection (10). Although attenuated live vaccines and nucleic acid vaccines of A. hydrophila also exhibit pretty good immune protective effects, these researches still remain in the laboratory stage (11)(12)(13).
However, due to the numerous serotypes of A. hydrophila and the wide range of pathogenic objects, it is difficult for inactivated vaccines to achieve broad-spectrum immune protection against A. hydrophila from different sources or serotypes. The various Omps of A. hydrophila with high immunogenicity are noteworthy (14), which can stimulate the humoral and cellular immunity of fish. Moreover, Omps are highly conserved among different strains (15), and are ideal antigens for preparing A. hydrophila subunit vaccines with broad-spectrum immune protective effects. The OmpA is a conserved antigen, which maintains the integrity of the outer membrane of A. hydrophila. The prepared OmpA antiserum of A. hydrophila J-1 strain isolated from Carassius auratus is broadspectrum, which shows strong immunoreaction with nine A. hydrophila strains of different serotypes (16). In addition, the OmpA subunit vaccine of A. hydrophila also shows excellent immune protective effects on I. punctatus (17). Therefore, the inactivated vaccines prepared by formalin inactivation and heat inactivation always exhibit excellent immune protective effects against specific A. hydrophila strain, but the broad-spectrum protective effects against various serotypes are limited. Usually, the protective effects and duration of protection of injection immunization are higher than that of immersion immunization for inactivated vaccines (13, 18). On the other hand, subunit vaccines prepared by isolation or recombinant expression of conserved antigens, usually show broad-spectrum immune protective effects against different serotypes of A. hydrophila. As the immunogenicity and stability of subunit vaccines are usually weaker than that of other types of vaccines, so injection immunization is a better choice to ensure excellent immune protective effects (19,20).
In the present study, we evaluated the protective effects of inactivated and OmpA subunit vaccines against A. hydrophila infection in juvenile M. amblycephala, and clarified whether the concentration of vaccines affected the immune protection. In addition, the immunity of M. amblycephala upon infection was detected by measuring the density of immune cells, the expression of immune genes, the titer of serum antibodies and the bactericidal ability. This study provides novel insights into the comparative evaluation of immunogenicity and protective effects of A. hydrophila inactivated vaccine and OmpA subunit vaccine, so as the theoretical basis for developing highefficiency aquatic vaccines.

Ethics statement
This study was approved by the Animal Care and Use Committee of Jiangsu Ocean University (protocol no. 2020-37, approval date: September 1, 2019). All procedures involving animals were performed in accordance with the guidelines for the Care and Use of Laboratory Animals in China.

A. hydrophila strain
The pathogenic A. hydrophila strain isolated from moribund M. amblycephala was stored at -80°C in our laboratory, which had been used in our previous studies (21). The stored A. hydrophila strain was cultured on Luria-Bertani (LB) agar plates, and single colonies of A. hydrophila were randomly isolated for expanding culture at 28°C for 16 h, which were then identified by PCR amplification and 16S rRNA sequencing (MAP, Shanghai, China). Then, the correctly identified A. hydrophila was stored at 4°C for further use.

Experimental fish
Healthy juvenile M. amblycephala (1.12 ± 0.22 g), obtained from a fish farm in Guangzhou, China, were confirmed free of infection by examining with standard bacteriological and parasitological examination (22). Experimental fish were fed with commercial feed for temporary rearing and taming for 2 weeks in an indoor freshwater recirculating system before the culture experiment.

Preparation of vaccines 2.4.1 A. hydrophila OmpA subunit vaccine
According to the genomic DNA sequence of A. hydrophila OmpA gene (GenBank accession number: AF146597), the primers were designed to construct the recombinant plasmid including the coding region of OmpA gene (Supplemental Table 1). The PCR amplified product was digested and cloned into pET-32a vector, and then transformed into Escherichia coli BL21 competent cells (TransGen Biotech, Beijing, China). The recombinant OmpA protein was induced by isopropyl-b-D-thiogalactopyranoside (IPTG) with a final concentration of 0.5 mM at 37°C for 10 h, which was purified using a Ni-Agarose His-tagged Protein Purification Kit (CoWin Biosciences, Jiangsu, China). Then, the protein concentration was detected by BCA Protein Assay Kit (CoWin Biosciences).

A. hydrophila inactivated vaccine
The identified A. hydrophila was incubated in LB liquid medium at 28°C for 24 h, followed by inactivated with 0.3% formaldehyde at 37°C for 16 h (23). Sterility assay was carried out by conventional plate culture method to ensure the safety of the prepared inactivated vaccine. Then, the inactivated A. hydrophila was collected by centrifuging at 3, 000 rpm for 10 min, and washed with phosphate buffered saline (PBS) for three times.

Safety detection of prepared vaccines
The A. hydrophila OmpA subunit vaccine and inactivated vaccine was mixed with MONTANIDE ™ ISA 763A VG adjuvant (Seppic, France) in the ratio of 3: 7, respectively. The final concentration of OmpA vaccine was set as 0.5 mg/mL and 1.0 mg/ mL according to previous studies with some adjustments (24-27), and the concentration of inactivated vaccine was 1×10 8 CFU/mL and 1×10 9 CFU/mL (10, 24), respectively. Juvenile M. amblycephala (n = 450) were randomly divided into 5 groups (90 fish per group with 3 tanks), and the emulsified vaccines were intraperitoneally injected with 20 mL for each fish, then the pathological symptoms or deaths were continuously recorded for 2 weeks.  Table 1, for the first immunization, the emulsions of mixed vaccines and adjuvant were intraperitoneally injected with 20 mL for each fish. Then, the booster immunization was conducted 2 weeks after the first vaccination, which was directly immunized with 20 mL PBS or vaccines without adjuvant.

Fish rearing and vaccination
The experimental fish were fed 4 times daily (8:00, 11:00, 14:00, and 17:00) to apparent satiation (approximately 3% of the body weight), and the water was renewed every day to maintain suitable water quality. The water temperature was maintained at 26-28°C with the dissolved oxygen greater than 6.0 mg/L, and the pH value was approximately 7.2, while ammonia, nitrogen, and nitrite were lower than 0.1 mg/L.

Bacterial challenge and sampling
The bacterial challenge assay was performed 2 weeks after the booster immunization as previously described (21), and experimental fish from each tank (60 fish/tank, 3 tanks/group) were assigned to 2 categories for calculating mortality (30 fish/tank) and sampling (30 fish/tank), respectively. The fish for sampling (body weight of 2.08 ± 0.25 g) were intraperitoneally injected with 20 mL of 1×10 7 CFU/mL A. hydrophila (LD50 dose). Then, 3 individuals from each tank were randomly dissected after anesthetized with MS-222, and the blood, gill, hepatopancreas and intestines were collected at 0, 1, 3, 7, 14 and 30 d post infection (dpi). The fish for calculating mortality (body weight of 2.08 ± 0.25 g) were intraperitoneally injected with 20 mL of 5×10 7 CFU/mL A. hydrophila, and the relative percent survival (RPS) was calculated using the formula: RPS = (1-% mortality in vaccinated fish/% mortality in control fish) × 100. Clinical signs and mortality were recorded everyday post infection for 14 d.
The blood was stored at 4°C overnight and centrifuged at 3000 rpm to isolate serum, which was used for Enzyme-linked immunosorbent assay (ELISA) assay and detection of enzymes activities, respectively. The hepatopancreas was homogenized and centrifuged at 4000 rpm for 10 min at 4°C for enzymes activities analysis. Immunohistochemical samples (gills, hepatopancreas and intestines) were fixed in 4% paraformaldehyde for 24 h at 4°C. Samples (gills, hepatopancreas and intestines) for RNA extraction were stored in sample protector for RNA (TaKaRa, Dalian, China) for 24 h at 4°C and then stored at -80°C until used.

Preparation and specificity verification of polyclonal antibodies
The recombinant IgM (AGR34023.1), IgD (AGR34025.1), IgZ (AGR34024.1) and CD8 (XP_048023282.1) protein were prepared in the same way as OmpA. After three immunizations with purified recombinant protein, rabbit antiserum against IgM, IgZ, IgD, and CD8 were obtained and purified using Protein A/G Agarose Kit (Beyotime, Shanghai, China). The total proteins of M. amblycephala tissues were extracted with RIPA lysate (Beyotime) and the concentration was determined with a BCA kit (Beyotime).

Enzyme-linked immunosorbent assay (ELISA)
The titer of serum immunoglobulin M (IgM) specific to A. hydrophila was detected by ELISA (29). In short, 96 well plates were coated with 200 mL of 5 mg/mL A. hydrophila ultrasonic fragments (same strain as used for inactivated vaccine preparation and challenge experiment) overnight at 4°C, and then blocked with 1% BSA at 22°C for 2 h, which were further incubated with M. amblycephala serum samples (1: 50 dilution) 96 well plates at 4°C for 12 h. Followed by incubating the 96 well plate with rabbit anti-M. amblycephala IgM specific antibody (1: 3000 dilution) at 22°C for 1 h, and incubating with goat anti-rabbit IgG-HRP secondary antibody (Beyotime; 1:2000 dilution) at 22°C for 1 h. After washing, the TMB substrate was added to incubate at 22°C for 5 min, then the termination solution was added to stop color reaction, and measured the absorbance at 450 nm. Internal positive and negative control samples were included. The

Hematoxylin and eosin (H&E) staining
Three fixed tissues from each group were dehydrated with gradient ethanol, cleaned in xylene substitute, embedded in paraffin blocks, sectioned at 4 mm thickness, and stained with H&E (Sigma, St. Louis, Missouri, USA) (21). Three random fields of each slide were captured (400×) using a light microscope (Nikon, Tokyo, Japan). Histopathological change scores were obtained by evaluating the degree of tissue lesion. The score of 0 means asymptomatic, and the higher the score, the more serious the lesion.

Immunohistochemistry assay (IHC)
The expression and distribution of immune-related proteins (CD8, IgM, IgZ, and IgD) were detected by immunohistochemistry assay as previously described (28). Briefly, after dehydration, transparency and penetration, samples were embedded in paraffin. Sections (4 mm) were prepared, deparaffinized and rehydrated, then endogenous peroxidase was removed by immersion in 3% hydrogen peroxide. Sections were incubated with primary antibody (1: 2000) overnight at 4°C and then incubated with goat anti-rabbit IgG-HRP secondary antibody at 37°C for 30 min. The immunoreaction products were visualized using a DAB kit, and sections were counterstained with hematoxylin and embedded in glycerol and then positive cells were counted in three random fields using a light microscope (Nikon) with magnifications (×400 for hepatopancreas and intestine). The density of positive cells per mm 2 was estimated as positive cells per mm 2 (total of positive cells/total area counted).

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNAs of collected samples were extracted using the RNA Easy Fast Tissue Kit (TIANGEN, Beijing, China) and the quality and concentration were determined by agarose gel electrophoresis and NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA), respectively. In addition, cDNA was synthesized using the PrimeScript ® RT reagent Kit with gDNA Eraser (TaKaRa) following the manufacturer's protocol.
The expression patterns of M. amblycephala immune-related genes (TNFa, iNOS, IL-1b, IL-6, IL-10, C3, SAA, TCR a, MHC I, MHC II, CD4, CD8, IgM, IgZ and IgD) and A. hydrophila 16S rRNA gene were analyzed by qRT-PCR, as previously reported (28). Briefly, qRT-PCR was performed on the ABI StepOne Plus real-time PCR system (PerkinElmer Applied Biosystems, CA, USA) using the QuantiNova ™ SYBR ® Green PCR Kit (TaKaRa). All the reactions were performed in triplicate with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene, and the primers are listed in Supplemental Table 1. The relative expression levels of target genes were measured in terms of the threshold cycle (Ct) value using the 2 -DDCt method (30). Gene expression levels of the control group were set as 1, and that of the vaccinated groups were presented as fold change. A. hydrophila abundance in different tissues of M. amblycephala from different groups was monitored by measuring A. hydrophila 16S rRNA transcripts levels, which was calculated according to the Ct values of qRT-PCR assay (29).

Analyses of antimicrobial enzymes activities
In this study, the activities of lysozyme (LZM), alkaline phosphatase (AKP), and acid phosphatase (ACP) in the serum and hepatopancreas were measured according to methods previously described (21). Briefly, according to the ratio of tissue weight (g) to phosphate buffer saline (PBS) volume (mL) of 1:9, hepatopancreas were homogenized. After centrifugation at 2500 rpm for 10 min, the supernatant was separated to determine the activities of antimicrobial enzymes. The activities of LZM, AKP and ACP were detected using the corresponding enzyme activity detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Statistical analysis
In the present study, data were presented as mean ± standard error (SE). The statistical significance was assessed by one-way analysis of variance (ANOVA), and multiple comparisons were performed using the Tukey method of SPSS 25.0. The P-value of 0.05 was considered as statistically significant difference.

Clinical symptoms and RPS
The recombinant OmpA protein was mainly expressed in the inclusion body with high purity (Figures 1A, B). The mortality rate in the control group was as high as 85.6% post A. hydrophila infection, while those of the inactivated vaccine L, inactivated vaccine H, OmpA vaccine L and OmpA vaccine H groups were 19.8%, 36%, 15% and 21%, respectively, which were significantly lower than that of the control group ( Figure 1C). In addition, the RPS values of inactivated vaccine L, inactivated vaccine H, OmpA vaccine L and OmpA vaccine H groups were 76.9%, 58%, 82.3% and 75.5%, respectively. These results indicated that prepared vaccines showed excellent immunoprotective effects, especially OmpA vaccine L group.
Obvious clinical symptoms were observed in the control group at 1 dpi, including congestion of fish body, hemorrhage and swelling around anus and intestines, as well as severe hemorrhage and necrosis of gills. At 3 dpi, control fish presented obvious congestion of gills, and the intestines became thinned, brittle and were easy to break. In contrast, the symptoms of vaccinated fish mainly included slight congestion of gills at 1-3 dpi, and the intestines became thinned and brittle in the inactivated vaccine groups at 1 dpi (Supplemental Figure 1). After 7 dpi, all the survived fish gradually recovered and the clinical symptoms disappeared. The record of clinical symptoms revealed the immunoprotective effect of A. hydrophila vaccine.

Inactivated and
OmpA subunit vaccines maintain the stability of histological structures of juvenile M. amblycephala

Gill
Histopathological changes of gill lamella congestion, necrosis, inflammatory cell infiltration and gill lamella hyperplasia were observed post infection. Three randomly selected fields (×400) were graded according to the histological changes (Table 2). Overall, hyperemia and inflammatory cell infiltration were found in gills lamellae of all fish at 1-7 dpi, while necrosis was commonly found in gills of control fish (Figure 2A). The gill lamella of all groups showed pathological characteristics at 1dpi. With the increase of infection time, the disease of gill lamella of immunized fish was alleviated and the tissue structure was improved, and the protective effect of OmpA vaccine was better than that of inactivated vaccine.

Hepatopancreas
Histopathological changes of hydropic degeneration, necrosis, sinusoidal congestion, inflammatory cell infiltration and eosinophilic staining were found in livers post infection. The severity of liver lesions was scored according to the histopathological changes as Table 2. Hydropic degeneration and focal necrosis were commonly observed in all livers at 1 dpi in both control and immunized groups. At 3 dpi, hydropic degeneration and rupture of hepatocytes leading to focal necrosis were evident in control livers, while the immunized livers displayed hepatocytes vacuolar degeneration and sinusoidal congestion. At 7 dpi, hepatocytes of control livers presented diffuse  necrosis, whereas only focal necrosis and sinusoidal congestion were observed ( Figure 2B).

Intestine
After bacterial infection, the major histopathological changes of intestine were intestinal villus necrosis (1 dpi) and submucosal edema (3 dpi) in control group, while the immunized intestine only displayed submucosa edema at 1 dpi (except for a small inflammatory cell infiltration at 7 dpi in inactivated vaccine H group) ( Figure 2C). The severity was scored according to the intestinal lesions as Table 2. The intestinal lesions of all groups were relatively high at 1dpi, which were significantly alleviated in the next few days in all immunized groups.

Inactivated and OmpA subunit vaccines increase the production of IgM specific to A. hydrophila
The specificity of prepared anti-IgM antibody and no cross reaction between IgD, IgZ and IgM was verified by western blotting (Supplemental Figures 2A, E). No significant difference in IgM levels between control and vaccinated groups before and at 7 dpi, with just slight fluctuations. At 14 dpi, the specific IgM levels in the vaccinated groups were up-regulated to the peak and higher than that of the control group, especially these of the two OmpA subunit vaccine groups were more significant (Figure 3), which indicated that vaccination increased the production of specific antibodies and enhanced host resistance to A. hydrophila infection.

Inactivated and OmpA subunit vaccines decrease the abundance of A. hydrophila in various tissues
The 16S rRNA transcripts levels of A. hydrophila in different tissues were detected to compare the bacterial abundance in different groups at various time points post infection. In the intestines, 16S rRNA transcripts of most vaccinated groups were lower than that of the control group, especially these of the OmpA vaccine groups at all time points. The OmpA vaccine groups also showed lower levels of 16S rRNA transcripts in the hepatopancreas and gills. However, the up-regulated levels of 16S rRNA transcripts were observed in the gills at 3 and 7 dpi of most groups, which should be related to the method of intraperitoneal injection. Overall, these results revealed that vaccines immunization (especially OmpA vaccines) decreased the A. hydrophila The histopathological changes post challenged by A hydroophila. Gills (A), asterisk: gill filament; black arrow: gill lamellae; blue arrow: respiratory epithelium slough off; rhombuses: gill filament edema; rectangle: lamellae necrosis. Hepatopancreas (B), asterisk: central vein; black arrow: hepatic sinusoid; blue arrow: congested central vein; yellow arrow: narrowed central vein, and intestines; triangle: degenerated hepatocytes; rectangle: focal necrosis. Intestine (C), rhombuses: submucosa; black arrow: intestinal villi; asterisk: submucosa edema; blue arrow: intestinal villi necrosis; yellow arrow: inflammatory cells. The specific IgM levels to A. hydrophila detected by ELISA. Asterisk (*) indicated significant difference between the vaccinated groups and control group (P < 0.05). The vertical cut-off line represented the control serum + 3×standard deviation.
abundance in the host, thereby protecting juvenile M. amblycephala against infection (Figure 4).

Inactivated and OmpA subunit vaccines increase immune cell quantity
The specificity of all prepared antibodies was validated by western blotting which showed excellent specificity (Supplemental Figure 2). Four immune-related proteins (CD8, IgM, IgZ and IgD) showed strong positive immunoreactive signals in both tissues (Table 3). In general, the number of immune positive cells was much more abundant in the hepatopancreas than in the intestines (except CD8 + cells), which also maintained abundant levels post infection, while that in the intestines just showed partial up-regulation post infection ( Figure 5).

CD8 a + cells
The CD8 a + cells were more abundant in vaccinated groups before infection (Supplemental Figures 3A, 4A, 5A) and significantly increased in all groups post infection, especially the control group that maintained at relatively high level. In general, whether challenged or not, the number and trend of CD8 a + cells FIGURE 4 Infection of M. amblycephala in experimental groups expressed as percentage of fish carrying A. hydrophila in different organs following challenge quantified by RT-qPCR with 16S rRNA. *: Significant difference compared to the control group at each time point (P < 0.05).
were consistent in vaccinated groups, which was more abundant in the high-dose vaccines groups.

IgD + cells
The IgD + cells were also more abundant in vaccinated groups before bacterial infection in the hepatopancreas and intestines (Supplemental Figures 3B, 4B, 5B), which also maintained abundant levels in the hepatopancreas of all groups post infection. In the intestines, the IgD + cells of control group only increased at 1 dpi, while it is more abundant in the OmpA vaccine immunized groups at 3 or 7 dpi.

IgM + cells
In general, the abundance of IgM + cells was much lower in both tissues at all time points compared to other immune cells (Supplemental Figures 3C, 4C, 5C). In addition, the number of IgM + cells was more abundant in the vaccinated groups compared with control group before infection and was not affected in control group post infection. Moreover, the abundance of IgM + cells was a bit up-regulated in the intestines of vaccinated groups post infection, while these in the hepatopancreas decreased.

IgZ + cells
In the hepatopancreas, the number of IgZ + cells was higher in immunized groups before infection, which also maintained abundant levels post infection, and the up-regulation in the control group was only observed at 7 dpi (Supplemental Figures 3D, 4D, 5D). Additionally, the abundance of IgZ + cells was generally lower in the intestines than that in the hepatopancreas, with only partial up-regulation found in the control and OmpA vaccine L groups at 1 dpi.

Inactivated and OmpA subunit vaccines promote the expression of immune related genes
In the intestines, the expression of adaptive immune-related genes (C3, TCR a, MHC I, MHC II, CD4, CD8, IgM, IgZ and IgD) was higher in immunized groups after 2 times vaccination, and most of them were also significantly up-regulated in the vaccinated groups post infection, except for CD4 and TCR a ( Figure 6A; Supplemental Figure 6A). In addition, proinflammatory cytokines The number of positive cells was calculated in an area of 1 mm 2 . Counting of each cell type was performed on 3 fish/group at 0, 1, 3 and 7 dpi. Red shading indicates a significant increase in positive cells, and green shading indicates a significant decrease in positive cells (P < 0.05). Intestine (I) and hepatopancreas (H).
(TNFa, iNOS, IL-1b, IL-6 and SAA) showed similar expression patterns to adaptive immune-related genes, especially the significant up-regulation of acute phase protein SAA, while the expression of anti-inflammatory cytokine IL-10 was mainly decreased post infection. These results indicated that vaccine immunization enhanced host innate and adaptive immunity in the intestines, especially the high dose vaccine groups.
In the gills, the expression of most adaptive immune-related genes was also higher in immunized groups after vaccination and post infection, except for MHC I ( Figure 6B; Supplemental Figure 6B). The expression of most proinflammatory cytokines was relatively low after vaccination, but up-regulated significantly at 3 and 7 dpi (SAA up-regulated significantly at 1 dpi). These results also revealed that vaccine immunization strengthened host innate and adaptive immunity in the gills.
In the hepatopancreas, similar expression patterns were found in adaptive and innate immune-related genes with low expression levels before 1 dpi, which started up-regulated from 3 dpi to 30 dpi (except for IgZ and MHC I). In addition, the vaccinated groups showed higher expression levels than those of the control group at most time points, indicating that vaccine immunization also enhanced host immunity ( Figure 6C; Supplemental Figure 6C).

Inactivated and OmpA subunit vaccines enhance the activities of antimicrobial enzymes
The activities of LZM, ACP and AKP in the hepatopancreas and serum of juvenile M. amblycephala post infection were detected to evaluate effects of vaccination on host antibacterial ability. In the serum, the LZM activities of most groups reached the peak levels at 1 dpi, and immunized groups showed much higher activities compared with control group (P < 0.05). In the hepatopancreas, all vaccinated groups showed much higher LZM activities at 0, 1 and 7 dpi. Although LZM activity of the control group increased significantly at 3 and 14 dpi, LZM activity of the OmpA vaccine groups was dramatically higher (P < 0.05). In general, the LZM activities of all groups were up-regulated post infection in hepatopancreas and serum ( Figures 7A, D), especially vaccinated groups, indicating that vaccine immunization enhanced the host antibacterial ability.
The activities of ACP showed different levels and trends in the hepatopancreas and serum, which maintained high and stable levels in the serum upon infection, and the control and vaccinated groups showed no significant difference with just a partial increase at 7 dpi ( Figures 7B, E). In contrast, the hepatic ACP activities of inactivated vaccine H and OmpA vaccine H groups were higher than that of the control group at 0 dpi, which gradually decreased post infection. Thus, these results indicated that the bactericidal effect of serum was more excellent than that of hepatopancreas, and vaccination significantly enhanced the bactericidal effect of serum post infection, but not that of hepatopancreas.
The activities of AKP were fluctuant within limits in both serum and hepatopancreas, except for the extremely significant upregulation of vaccinated groups in the hepatopancreas at 0 dpi, but the regulation of vaccination was also inconsistent at various time points post infection, indicating that the variable effects of vaccination on inflammatory response upon infection. In a word, vaccines immunization (especially OmpA vaccines) enhanced the antibacterial ability of juvenile M. amblycephala against A. hydrophila infection (Figures 7C, F).

Discussion
Vaccine immunization is an important method for disease prevention and control in aquaculture, and more than 140 kinds of aquatic vaccines have been approved for marketing in the world (31). The inactivated A. hydrophila vaccines always provide excellent immune protective effects by inducing immune responses of aquatic animals, which thereby were widely studied and applied (32). However, due to wide range of sources and serotypes of A. hydrophila, which limit the broad-spectrum immune protective effects and application scopes of inactivated A. hydrophila vaccines. Studies have shown that the outer membrane proteins of A. hydrophila have high immunogenicity, which can stimulate the humoral and cellular immunity of fish. In addition, FIGURE 5 Heatmap illustrates results from IHC assay for the number of immune positive cells that affected by vaccination in the hepatopancreas and intestines at different time points post infection. Color value: log2 (fold change). they are highly conserved among different strains, thus are ideal antigens for preparing A. hydrophila vaccines with broad-spectrum immune protection (14,15).
The immunized recombinant Aha1 and OmpW proteins show significant protective effects on the carp upon A. hydrophila infection with the production of specific antibodies (14). Similarly, oral immunization of rOmpW protein also shows protective immunity in a dose-dependent manner (33). Indian carp immunized with OmpC subunit vaccine also achieves immune protective effect, and detectable cross-reacts of rOmpC antiserum with different Aeromonas lysates (34). In addition, other studies have evaluated the high immunogenicity of A. hydrophila OMPs, including Omp38 (24), OmpA1 (26), OmpF (35), Aha1 (36). In this study, we evaluated the relative survival rate of the prepared A. hydrophila inactivated vaccine and OmpA subunit vaccine upon bacterial infection, and both vaccines exhibited excellent immune protection to the juvenile B C A FIGURE 6 Heatmaps illustrate results from qRT-PCR for the expression of immune related genes in the intestines (A), gills (B) and hepatopancreas (C) of juvenile M. amblycephala. Color value: log2 (fold change).
M. amblycephala, among which the OmpA subunit vaccine showed better immune protective effect than that of the inactivated vaccine.
The protective effect of A. hydrophila inactivated vaccine and OmpA subunit vaccine should be correlated with the increased production of specific antibody, the decreased abundance of pathogen in the host, the enhanced expression and distribution of immune genes, improved histological structures and activities of antibacterial enzymes in M. amblycephala. In this study, the vaccines immunization could effectively reduce the pathological characteristics and damage of tissues caused by A. hydrophila infection and maintain the integrity of tissues, thereby protecting the host from inflammatory damage, which has also been observed in Cirrhinus mrigala and C. idella (37). Serum immunoglobulin IgM is the main antibody produced fish post infection or pathogenic antigen stimulation, which has the functions of dissolving bacteria, activating complements, immune conditioning and agglutination, and also plays a role in clearing pathogens at the early stage of infection. In this study, the up-regulated production of specific antibody against A. hydrophila in the vaccinated groups (especially OmpA vaccines) could enhance the host adaptive immunity, thereby decreasing the cumulative mortality post bacterial infection and exhibiting excellent immune protective effects, which is consistent with the studies in I. punctatus and P. mesopotamicus (10,26). Dietary probiotics as an immunomodulator can also increase the level of IgM in the serum of Mycteroperca rosacea (38). Similarly, when the content of arginine in feed increases, the production of IgM is accelerated, thus the disease resistance of experimental fish is improved (39).
Previous study has revealed that the mean bacterial concentrations in immune tissues were significantly lower in I. punctatus vaccinated with the fimbrial or OmpA1 than in the nonvaccinated control group (26,40). Similarly, the present study found the abundance of A. hydrophila in the tissues of juvenile M. amblycephala was affected by vaccination, which was significantly down-regulated in the vaccines immunized groups, especially that of the OmpA vaccine groups, indicating that vaccines immunization could reduce the proliferation of A. hydrophila in the host and thereby protecting host from inflammatory damage and death.
The specific immune response is usually studied to determine the effect of vaccine immunization on bacteria-infected organisms, but some studies have shown that vaccination also has a positive effect on the non-specific immune response, which is also an important defense mechanism to protect fish from bacterial infection. Antimicrobial enzymes such as LZM, ACP and AKP are common indicators for evaluating host nonspecific immunity (41). LZM has high bactericidal or hemolytic activity against pathogens, and ACP and AKP may contribute to promoting immunogenic responses (42)(43)(44), which thereby play vital roles in host defense system. In the present study, the levels of LZM, ACP and AKP in vaccinated groups were up-regulated, which was similar to the results in C. carpio with A. hydrophila Aha1 vaccine immunization and other previous studies, indicating that both OmpA subunit vaccine and inactivated vaccine can improve host non-specific immunity and enhance their ability to resist infection (10,36).
It has been proved that hepatopancreas, intestines and gills are important immune tissues in fish, which participate in host immunity, thereby immune related genes in these tissues play important roles in the immune defense responses post bacterial infection. The present study revealed that the number of CD8, IgD, IgZ, and IgM positive cells in the intestine and hepatopancreas increased post infection, especially that of the vaccinated groups, indicating that vaccines immunization activated host adaptive immune system and thus enhanced bacterial clearance ability. Consistently, the present study found that the expression of adaptive immune-related genes (C3, TCR a, MHC I, MHC II, CD4, CD8, IgM, IgZ and IgD) were higher in the vaccines immunized groups after 2 times vaccination, and most of them were also significantly up-regulated in the vaccinated groups post infection. Similar expression patterns of MHC I and MHC II genes were also observed in Pseudosciaena crocea (45), Salmo salar (46) and M. amblycephala (47).
CD8 + and CD4 + T cells use the T cell receptors (TCRs) to recognize complexes of pathogen-specific peptides antigens that are presented by major histocompatibility (MHC) class I and class II molecules on the surface of infected cells, respectively (48). Therefore, CD4 and CD8 genes are up-regulated in the immunerelated tissues of the juvenile M. amblycephala post infection, which might interact with MHC class II or I molecule and play an important role in the immune response. In previous study, CD8 + T cells play a role in the adaptive immunity of C. auratus against parasites infection, especially in the kidney and gills (49). It was also observed that the leukocytes of fish infected with virus showed high level of cytotoxicity and abundantly expressed CD8 a and TCR b, exerting specific immune functions (50). Immunoglobulins (Igs) are important molecules in fish adaptive immune system with high specificity, which were up-regulated when M. amblycephala infected by A. hydrophila (51-53). Consistent with this study, C. idella IgM gene expression was also induced after vaccination of single-walled carbon nanotubes (SWCNTs) coated A. hydrophila aerA subunit vaccine (25).
In the present study, expression of inflammatory cytokines (IL-1 b, IL-6, IL-10, TNF a and iNOS) and acute phase protein SAA were up-regulated post infection, especially that of the vaccines immunized groups, indicating that vaccination promoted host innate immunity of M. amblycephala. Similarly, the recombinant A. hydrophila OmpF vaccine immunized Labeo rohita also showed induced the expression of the immune-related genes such as IL-1 b and TNF a in the head kidney tissues, when compared to the control group at different time points post vaccination (35). These studies revealed that A. hydrophila vaccines immunization could enhance the innate immunity of fish, thus improving host resistance to bacterial infection.

Conclusions
Herein, we comparatively evaluated the immunogenicity and protective effects of A. hydrophila inactivated vaccine and OmpA subunit vaccine in M. amblycephala for the first time, and presented more comprehensive evaluation index. This study revealed that both vaccines (especially OmpA subunit vaccine) exhibited excellent protective effects on juvenile M. amblycephala, which should attribute to the increased production of specific antibody, and the decreased abundance of A. hydrophila in the tissues, and the enhanced expression of immune-related genes, and the improved histological structures and antibacterial enzymes activities. Therefore, both A. hydrophila inactivated vaccine and OmpA subunit vaccine are efficient vaccines, while OmpA subunit vaccine is a more ideal candidate vaccine against A. hydrophila.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethics statement
The animal study was reviewed and approved by the Animal Care and Use Committee of Jiangsu Ocean University.