Pathogenicity of Aeromonas veronii Causing Mass Mortality of Largemouth Bass (Micropterus salmoides) and Its Induced Host Immune Response

Aeromonas veronii is as an important opportunist pathogen of many aquatic animals, which is wildly distributed in various aquatic environments. In this study, a dominant bacterium GJL1 isolated from diseased M. salmoides was identified as A. veronii according to the morphological, physiological, and biochemical characteristics, as well as molecular identification. Detection of the virulence genes showed the isolate GJL1 carried outer membrane protein A (ompA), flagellin (flgA, flgM, flgN), aerolysin (aer), cytolytic enterotoxin (act), DNases (exu), and hemolysin (hly), and the isolate GJL1 also produced caseinase, lipase, gelatinase, and hemolysin. The virulence of strain GJL1 was confirmed by experimental infection; the median lethal dosage (LD50) of the GJL1 for largemouth bass was 3.6 × 105 CFU/mL, and histopathological analysis revealed that the isolate could cause obvious inflammatory responses in M. salmoides. Additionally, the immune-related gene expression in M. salmoides was evaluated, and the results showed that IgM, HIF-1α, Hep-1, IL-15, TGF-β1, and Cas-3 were significantly upregulated after A. veronii infection. Our results indicated that A. veronii was an etiological agent causing the mass mortality of M. salmoides, which contributes to understanding the immune response of M. salmoides against A. veronii infection.


Introduction
As an economically significant aquatic species native to North America, M. salmoides has been widely cultured in China [1], and the annual production has exceeded 619 thousand tons, according to the China Fishery Statistical Yearbook in 2020. Unfortunately, M. salmoides has suffered from increasing diseases due to the high-density culture and the deterioration of the water environment. In recent years, various viral pathogens have been reported to cause serious economic losses to the M. salmoides industry, including largemouth bass virus (LMBV), largemouth bass Birnavirus (LBBV), viral hemorrhagic septicemia virus (VHSV), nervous necrosis virus (NNV), and Micropterus salmoides rhabdovirus (MSRV) [2][3][4][5][6]. In addition, outbreaks caused by bacterial pathogens including Aeromonas hydrophila, A. veronii, Aeromonas sobria, Vibrio parahemolyticus, Nocardia seriolae, Edwardsiella piscicida, and Francisella orientalis, are also increasing in frequency and causing major economic losses [7][8][9][10][11][12][13]. In this study, the mass mortality of M. salmoides with skin ulcerations occurred in Yangzhou, Jiangsu Province, and the dominant bacterium GJL1 from the diseased M. salmoides was identified as A. veronii.
A. veronii, a Gram-negative bacterium, is widely distributed in freshwater and estuary environments and is an opportunistic pathogenic bacterium, which infects a variety of aquatic organisms. In recent years, A. veronii has been recognized as an aquatic pathogen for various fish species, such as Ictalurus punctatus, Oreochromis niloticus, Dicentrachus labrax,

Bacterial Virulence Assay
The isolate GJL1, as a representative of the dominant strains, was incubated in an LB medium at 28 • C with shaking at 180 rpm for 18 h, and the bacterial suspension was diluted from 2.4 × 10 8 to 2.4 × 10 5 CFU/mL by sterile PBS. Twenty healthy M. salmoides (60-70 g) in each tank (in triplicate) were injected intraperitoneally with 100 µL with different concentrations of the bacterial suspension (2.4 × 10 8 , 2.4 × 10 7 , 2.4 × 10 6 , and 2.4 × 10 5 CFU/mL) per fish, respectively, and the fish in the control group were injected with 100 µL sterile PBS (pH 7.4). The mortalities of fish were monitored every day for 14 d, and the LD 50 of A. veronii to M. salmoides was calculated based on the cumulative mortality of the fish using the methods of Behreans and Karber [32].

Histopathology
The livers, spleens, kidneys, and gills from the infected and control groups were fixed in Bouin's fixative, dehydrated in different concentrations of ethanol, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin (H&E) for histological examination.

Morphology Observation
The isolate GJL1 was observed under transmission electron microscopy (Tecnai 12, Philips, Eindhoven, The Netherlands). Briefly, the cells were harvested by centrifugation (4000 rpm, 15 min, 4 • C) and washed thrice with sterilized PBS (pH 7.4). Then, the cells were fixed in 2.5% glutaraldehyde, post-fixed with osmium tetroxide, dehydrated by a graded ethanol series, and coated with gold palladium alloy. Finally, the cells were observed with a transmission electron microscope, and the types and sizes of flagella were analyzed.
The 16S rRNA and gyrB genes of the isolate GJL1 were amplified as described by Zhang et al. [34]. After sequencing, the 16S rRNA and gyrB sequences of isolate GJL1 were searched in the NCBI database for sequence homology analysis using BLAST, and phylogenetic trees were constructed using the maximum likelihood method by MEGA 7.0 (version 7.0, Mega Limited, Auckland, New Zealand) [35].

Determination of Extracellular Enzymes and Hemolysin
The isolated A. veronii was screened for extracellular enzymatic activities, such as phospholipase, lipase, amylase, hemolysin, and urease, which were determined by the method described earlier by Gao et al. [30]. LB nutrient agar medium was supplemented with 7% rabbit erythrocytes, 2% starch, 1% gelatin, 1% Tween-80, and 10% egg yolk, respectively. Five microliters of a suspension of GJL1 were spot-inoculated in the center of the plates, which were incubated at 28 • C for 24 h. The presence of a lytic halo surrounding the GJL1 colonies was observed. The test was performed in triplicate.

Detection of the Expression Levels of Immune-Related Genes
The expression of immune-related genes (IgM, HIF-1α, Hep-1, IL-15, TGF-β1, and Cas-3) in the tissues of M. salmoides was monitored after A. veronii infection by using qRT-PCR. Briefly, a total of 40 fish were intraperitoneally injected with 100 µL A. veronii (3.6 × 10 5 CFU/mL), and the fish in the control group were injected with 100 µL sterile PBS. The liver, spleen, and kidney were sampled at 6, 12, 24, 48, and 72 h post infection. The qRT-PCR reactions were performed using Thermofisher QuantStudio Real-Time PCR System PCR System with a ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing Co., Ltd., Nanjing, China), and the primer sequences are displayed in Table S2. β-actin was chosen as an internal control, and the relative mRNA expression was calculated by the 2 −∆∆Ct method. The significant differences were analyzed by a t test using SPSS 16.0 software (p < 0.05). All qRT-PCR reactions were performed in triplicate.

Pathological Symptoms
The epidemiological investigation found that the diseased M. salmoides showed serious ulceration on the surface, with hemorrhage in the bodies. The diseased fish had several common symptoms such as swelling and hemorrhage on the base of internal organs.

Isolation of Bacteria from Diseased M. salmoides
The pathological tissues of the diseased M. salmoides were isolated with abundant pure bacteria from the livers, spleens, kidneys, and gill samples, and these colonies grew with the characteristics of white color, translucence, circularity, convexity, and an intact edge. Pure isolates were obtained by streaking the colonies on LB nutrient agar plates, and a representative strain from these was chosen for this study, which was tentatively named GJL1.

Virulence of the Isolate
The results of the pathogenicity study are shown in Figure 1. The infected M. salmoide started to die from day 2, the 1.8 × 10 8 , 1.8 × 10 7 , 1.8 × 10 6 , and 1.8 × 10 5 CFU/ mL of GJL1 caused 100%, 80%, 40%, and 20% mortality after 14 dpi, respectively, and no fish died in the control group. The calculated LD 50 of GJL1 to the M. salmoides was 3.6 × 10 5 CFU/mL. Furthermore, the isolate GJL1 was reisolated from the infected M. salmoides, confirming that the experiment fulfilled Koch's postulates.
The pathological tissues of the diseased M. salmoides were isolated with abundant pure bacteria from the livers, spleens, kidneys, and gill samples, and these colonies grew with the characteristics of white color, translucence, circularity, convexity, and an intact edge. Pure isolates were obtained by streaking the colonies on LB nutrient agar plates, and a representative strain from these was chosen for this study, which was tentatively named GJL1.

Virulence of the Isolate
The results of the pathogenicity study are shown in Figure 1. The infected M. salmoide started to die from day 2, the 1.8 × 10 8 , 1.8 × 10 7 , 1.8 × 10 6 , and 1.8 × 10 5 CFU/ mL of GJL1 caused 100%, 80%, 40%, and 20% mortality after 14 dpi, respectively, and no fish died in the control group. The calculated LD50 of GJL1 to the M. salmoides was 3.6 × 10 5 CFU/mL. Furthermore, the isolate GJL1 was reisolated from the infected M. salmoides, confirming that the experiment fulfilled Koch's postulates.

Histological Observation
Compared with the control group, histopathologic examination showed hemorrhage and necrosis in liver tissues and the destruction of intercellular junctions between liver cells ( Figure 2B). As shown in Figure 2D, the spleen tissues showed several signs of telangiectasia, hyperemia, hemolysis, and the formation of blood spots, especially with severe regional rupture. Obvious signs of necrosis in the respiratory epithelial cells of the secondary gill plate were observed, and the gill lamellae were arranged irregularly, bent, and wrinkled. As shown in Figure 2H, nephritis occurred in the focal area of the kidney, the glomerulus necrosed, and the interrenal tissue cells were necrotic and chapped.

Histological Observation
Compared with the control group, histopathologic examination showed hemorrhage and necrosis in liver tissues and the destruction of intercellular junctions between liver cells ( Figure 2B). As shown in Figure 2D, the spleen tissues showed several signs of telangiectasia, hyperemia, hemolysis, and the formation of blood spots, especially with severe regional rupture. Obvious signs of necrosis in the respiratory epithelial cells of the secondary gill plate were observed, and the gill lamellae were arranged irregularly, bent, and wrinkled. As shown in Figure 2H, nephritis occurred in the focal area of the kidney, the glomerulus necrosed, and the interrenal tissue cells were necrotic and chapped. (G) histologic section of healthy kidney; (H) histologic section of infected kidney. LD represents decreased lipid droplets; HC represents mild hepatic cell; CV represents swollen central vein. WP represents white pulp; RP represents red pulp. H represents hypertrophy; HP represents hyperplasia; ED represents epithelial cell detachment. G represents glomerulus; UT represents urine tubules.

Electron Microscopic Observation of the Isolate
The micrographs of transmission electron microscopy revealed that the isolate GJL1 was rod-shaped with round-ends, approximately 1.1-1.9 µm wide and 2.6-4.8 µm long, which was motile by single polar flagella ( Figure 3).

Physiological and Biochemical Characterization
The isolate GJL1 was obtained from the diseased sample M. salmoides and confirmed as A. veronii bv veronii by morphological, physiological, and biochemical characteristics as described in Bergey's Manual of Systematic Bacteriology. As shown in Table 1, the motility, indole, sucrose, salicin, α-Methyl-d-glucoside, esculin hydrolysis, and ornithine decarboxylase were positive but not arginine dihydrolase. The Voges-Proskauer, raffinose, β-galactosidase, dulcitol and fructose activity of the isolate GJL1 were positive, which showed different characteristics than the descriptions of A. veronii in Bergey's Manual of Systematic Bacteriology.

Physiological and Biochemical Characterization
The isolate GJL1 was obtained from the diseased sample M. salmoides and confirmed as A. veronii bv veronii by morphological, physiological, and biochemical characteristics as described in Bergey's Manual of Systematic Bacteriology. As shown in Table 1, the motility, indole, sucrose, salicin, α-Methyl-d-glucoside, esculin hydrolysis, and ornithine decarboxylase were positive but not arginine dihydrolase. The Voges-Proskauer, raffinose, β-galactosidase, dulcitol and fructose activity of the isolate GJL1 were positive, which showed different characteristics than the descriptions of A. veronii in Bergey's Manual of Systematic Bacteriology.

Virulence Factors and Genes of the Pathogenic Isolate
The extracellular enzymes activities of GJL1 are shown in Figure 4. The strain GJL1 produced DNAase, protease, gelatinase, and hemolysin activity, without lecithin and lipase activity ( Figure 5). The extracellular enzymes activities of GJL1 are shown in Figure 4. The strain GJL1 produced DNAase, protease, gelatinase, and hemolysin activity, without lecithin and lipase activity ( Figure 5).

. Immune-Related Gene Expression in Livers at Different Hours Post-Infection
As shown in Figure 7, significant expression levels of IgM, HIF-1α, Hep-1, IL-15, TGF-β1, and Cas-3 were detected at 12 hpi. Then, the increased rates of IgM, IL-15, and Cas-3 were reduced between 12 hpi and 48 hpi, and infected group remained higher than the control group, except for IL-15 and Cas-3. The expression peaks of IgM, HIF-1α, IL-15, and Cas-3 in the liver were at 12 hpi, and reached 1.91-, 2.80-, 3.60-, and 1.40-fold, respectively. The expression peak of TGF-β1 in the liver was at 24 hpi and reached 2.23-fold. The expression level of Hep-1 in the liver reached the peak value of 2.39-fold at 72 hpi.

Immune-Related Gene Expression in Spleens at Different Hours Post-Infection
As shown in Figure 8, the significant expression levels of IgM, HIF-1α, Hep-1, IL-15, TGF-β1, and Cas-3 were all detected at different times. The increased rates of IgM and IL-15 were reduced between 12 hpi and 48 hpi, and the infected group remained higher than the control group. The expression peaks of IgM and IL-15 in the spleen were at 24 hpi and reached 1.93-and 1.73-fold, respectively. The expression peak of TGF-β1 in the spleen was at 48 hpi and reached 2.57-fold. The expression levels of HIF-1α, Hep-1, and Cas-3 in the spleen reached the peak values of 3.15-, 2.03-, and 4.40-fold higher, respectively, at 72 hpi.

Discussion
A. veronii causes one of the most common conditional pathogens of freshwater fish cultured in China and has been known to cause significant economic damage in the aquaculture industry [36]. The cases of death caused by A. veronii have risen quickly in recent

Discussion
A. veronii causes one of the most common conditional pathogens of freshwater fish cultured in China and has been known to cause significant economic damage in the aquaculture industry [36]. The cases of death caused by A. veronii have risen quickly in recent

Discussion
A. veronii causes one of the most common conditional pathogens of freshwater fish cultured in China and has been known to cause significant economic damage in the aquaculture industry [36]. The cases of death caused by A. veronii have risen quickly in recent years, with the pathological symptoms in fish including skin ulcers, bleeding of organs, and severe ascites. Shameena et al. indicated that A. veronii isolated from diseased C. auratus caused high economic losses in farming [25]. Hoai et al. reported the disease and mortality of channel catfish mainly due to A. veronii [19]. In addition, A. veronii was also pathogenic to Poecilia reticulata [37]. In this study, A. veronii GJL1 was isolated from diseased M. salmoides showing serious ulceration on the surface and hemorrhage in the bodies. Challenge tests showed that the LD 50 of A. veronii GJL1 to M. salmoides was 3.6 × 10 5 CFU/mL, and the challenged M. salmoides exhibited similar symptoms to the naturally infected fish, suggesting that the isolate GJL1 has high virulence to M. salmoides.
Previous studies have shown that extracellular products of bacteria are considered as important factors in the infection of the host. It is reported that many virulence factors, such as amylase, caseinase, gelatinase, lipase, hemolysin, and aerolysin, play important roles in the pathogenicity of A. veronii. [38,39]. In the present study, the isolate GJL1 exhibited caseinase, lipase, gelatinase, and hemolysin activities, which contributed to invading the host. Further, the virulence-related genes encode secreted proteins and toxins that may play important roles in the pathogenesis of A. veronii. Sreedharan et al. reported that various virulence genes, such as act and alt coding enterotoxins, aerA coding enterotoxins, and hlyA coding hemolytic toxins, etc., were key contributors to the virulence of A. veronii [40]. Moreover, the aer gene was an important gene associated with aerolysin [41]. Gao et al. reported that the expression of hly could cause cytotoxic effects and the lysis of erythrocytes [42]. Meanwhile, the fla gene plays an important role in the abilities of motility and adherence to cells [43]. In this study, the virulence-related genes including ompA, flgA, flgM, flgN, aer, act, exu, and hly were detected in A. veronii GJL1. These results indicated that the highly virulent A. veronii GJL1 may harbor many virulence genes.
Fish possess an adaptive immune system with an ability to mount a specific antibody response against pathogens, and various aspects of the innate immune systems and tissues have been studied in M. salmoides. In this study, the expressions of six immune-related genes in M. salmoides were determined after A. veronii infection, which exhibited significantly differential expressions. Transforming growth factor-β (TGF-β) is an anti-inflammatory cytokine, and TGF-β1 is an important isoform of TGF-β, which has been proved to relate to the controlled inflammation by interleukin [44,45]. IL15 plays an important role in innate and adaptive immunity, which is one of the most important factors to regulate T-cell, dendritic cell, and NK cell development and participate in some immune related signal transduction pathways [46]. The signaling molecules involved in mediating IL-15induced B cell activation were identified that culminated in augmenting IgM response [47]. Meanwhile, as the systemic immunoglobulin, IgM is not only the major antibody of primary response but also a vital part of the adaptive immune response of fish [48]. Hypoxiainducible factor (HIF) can induce apoptosis to release inflammatory mediators such as IL-1β and TNF-α [49]. The expression of hepcidin was also shown to be positively regulated by TGF-β /SMAD4 signals [50]. In addition, Caspase-3 is the key executory enzyme and final effector of apoptosis [51]. The activation level of caspase-3 was surveyed to understand the apoptosis status of the liver and spleen in largemouth bass during bacterial infection. In this study, the expression levels of the above six immune-related genes of M. salmoides infected by A. veronii were studied; the expression of IgM was significantly upregulated from 6 to 24 hpi in the liver and spleen, and the HIF-1α, Hep-1, and TGF-β1 expression levels in the liver and spleen were also significantly upregulated after A. veronii infection. In addition, the expression levels of IL-15 and Cas-3 in the liver were found to reach the maximum at 12 hpi but were rapidly downregulated after 24 hpi. Our results revealed that these immune-related genes were influenced by A. veronii and activated the host immune defense system, which provides a theoretical basis of the M. salmoides and A. veronii interactions.
In conclusion, the A. veronii GJL1 was identified as highly pathogenic to M. salmoides in this study. The expression levels of the immune-related genes, including IgM, HIF-1α, Hep-1, IL-15, TGF-β1, and Cas-3, of M. salmoides were significantly changed during the time course of the immune response to the pathogenic A. veronii. Furthermore, these findings provide theoretical support for prevention and control of the diseases caused by A. veronii in aquaculture.