Isolation, Identiﬁcation and Characteristics of Aeromonas caviae from Diseased Largemouth Bass ( Micropterus salmoides )

: The largemouth bass ( Micropterus salmoides ) is one of the most economically valuable ﬁsh species in China. In this study, a bacterial pathogen was isolated from the internal organs of diseased M. salmoides , and the strain was named WH21406. This isolate was identiﬁed as Aeromonas caviae on the basis of its morphology, biochemical features and 16S rDNA phylogenetic analysis. Four virulence genes related to pathogenicity, namely, ﬂagella ( ﬂa ), elastase ( ela ), haemolysin ( hly ) and aerolysin ( aer ), were detected in this isolate. The median lethal dosage (LD50) of A. caviae WH21406 for M. salmoides was calculated to be 3.46 × 10 5 CFU mL − 1 . The histopathological analysis showed obvious tissue damage in the gill, liver, kidney, spleen and gut of the diseased ﬁsh. The antibiotic susceptibility test demonstrated that strain WH21406 was highly sensitive to enroﬂoxacin, norﬂoxacin, streptomycin and amikacin. The results of this study provide a foundation for the diagnosis, prevention and treatment of A. caviae infection in M. salmoides . Author Contributions: Conceptualization, M.X. and Y.Z.; methodology, M.X.; software, Z.X.; vali-dation, Y.L., N.J. and M.X.; formal analysis, W.L.; investigation, Y.M.; resources, Z.X.; data curation, M.X.; writing—original draft preparation, M.X.; writing—review and editing, Y.Z.; visualization, N.J.; supervision, L.Z.; project administration, Y.F.; funding acquisition, Y.Z. All authors have read to the published version of the manuscript.


Introduction
The largemouth bass (Micropterus salmoides) is indigenous to the Mississippi River Valley in North America, and it was introduced to various countries, including China [1,2]. Owing to its rapid growth and delicious flesh, M. salmoides has become one of the most economically valuable fish species in China [3][4][5]. According to the China Fishery Statistical Yearbook, the annual output of M. salmoides already reached 619,519 tons in 2020 (Fisheries and Fisheries Administration Bureau of Ministry of Agriculture and Rural Affairs, 2021). However, the aquatic environment is deteriorating as a result of the rapid expansion of largemouth bass aquaculture, resulting in increasing incidences of serious pathogen outbreaks that threaten the sustainable development of largemouth bass aquaculture [3,6,7]. Outbreaks caused by bacterial pathogens such as Nocardia seriolae, Aeromonas hydrophila, Aeromonas veronii, Aeromonas sobria, Edwardsiella piscicida, Edwarsiella tarda and Flavobacterium columnare are increasing in frequency and causing huge economic losses [8][9][10][11][12][13][14].
The pathogenicity of Aeromonas species is complex and confounding. Aeromonas produces various toxins that can harm its hosts such as hemolysin, aerolysin and cytotonic enterotoxins [25]. Dallal et al. found the presence of 5 virulence genes such as alt, ast, act, aer and hly among 12 strains of Aeromonas isolates, including A. veronii, A. hydrophila, and A. caviae [26]. Hence, it is imperative to detect the virulence genes in Aeromonas in order to assess its potential pathogenicity.
To the best of our knowledge, this is the first report of A. caviae as a causative agent in the largemouth bass (M. salmoides). The diseased M. salmoides typically showed anabrosis and redness of the body surface, and a large number of deaths were detected. The present study aimed to investigate the etiology of this disease and provide scientific reference for its diagnosis and treatment. An A. caviae strain was isolated from the diseased fish and identified through morphological observation, bacterial biochemical identification and 16S rDNA sequence analysis. The pathogenicity of this A. caviae strain was confirmed. In addition, histopathological observation of the diseased fish, virulent gene analysis and antibiotic susceptibility screening were performed to obtain information for the healthy breeding of M. salmoides.

Fish Specimens
Diseased M. salmoides specimens (15 ± 2 cm in length) were collected from the M. salmoides breeding base in Wuhan, Hubei Province, China. The diseased M. salmoides showed reduced feeding and slow swimming. The clinical symptoms of diseased M. salmoides included lethargy, surface swimming, redness of the skin and mouth, ulcers and turgidity. The illness of M. salmoides was lasted for 10 days, and the condition was controlled after drug treatment. The moribund fish were maintained in oxygenated bags and immediately transported to the laboratory for diagnosis and pathogen isolation. Healthy M. salmoides specimens (15 ± 2 cm in length) with no history of disease were obtained from the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. The fish were maintained in recirculating aquaria (300 L) for 14 days to allow them to acclimatize to the environment. During the acclimatization period, the water temperature was 28 • C ± 1 • C, the fish were fed with commercial feed twice a day and the water was renewed daily at a rate of 30%. All experimental procedures were conducted according to the guidelines of the Animal Experimental Ethical Inspection of Laboratory Animal Centre, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences (ID Number: YFI2021-zhouyong-05).

Pathogen Detection and Identification
Samples of the gills, skin mucus, liver, kidney and spleen were subjected to standardized virological and parasitological analyses for diagnostic purposes. The fish liver, spleen and kidney were homogenized and filtered with 0.22 µm micron membrane for virus examination. The liver cell line of M. salmoides was used for virus isolation. The parasites were detected in diseased fish using an inverted microscope. Bacterial isolation was performed in a class II biosafety cabinet (ESCO, Singapore). The moribund fish were anesthetized in 40 mg L −1 tricaine methane sulfonate (MS-222; Sigma, Saint Louis, MO, USA), and the body surface was disinfected before dissection. The liver and kidney of each moribund fish were removed with an inoculation loop, inoculated onto brain heart infusion (BHI) agar plates (Difco, Detroit, MI, USA) and incubated at 28 • C for 24 h. The colonies were subcultured twice, inoculated into BHI liquid medium and cultured at 28 • C with shaking at 200 rpm. Purified bacterial cultures were used for Gram staining (Jiancheng, Nanjing, China) and observed with a scanning electron microscope (Hitachi, Tokyo, Japan). The obtained strain was designated WH21406.

16S Ribosomal DNA Sequencing Analysis
The genomic DNA of WH21406 was extracted using a bacterial genomic DNA kit (Tiangen, Beijing, China). Universal primers 27F and 1492R (Table 1) were used to amplify the 16S rDNA. The amplification program was as follows: 95 • C for 5 min, followed by 35 cycles of 94 • C for 1 min, 55 • C for 1 min and 72 • C for 1 min and 72 • C for 10 min. The amplified products were analysed using 1% agarose gel electrophoresis and visualized with an ultraviolet light transilluminator (Bio-Rad, Hercules, CA, USA). The amplified products were collected and sent to Huayu Gene Technology Co., Ltd., Wuhan, China, for sequencing; the results were compared against the NCBI database (http://blast.ncbi.nlm.nih.gov, accessed on 19 May 2022).

Bacterial Biochemical Identification
The bacterial biochemical characteristics were examined using a Biolog automatic microbial identification system (Biolog, Hayward, CA, USA). The bacterial strains were inoculated onto BUG solid medium at 30 • C for 24 h. A single colony was selected and evenly distributed in IF-A inoculation solution, which was then inoculated into a GEN III identification plate at a dose of 100 µL per well. The GEN III identification plate was placed in the Biolog automatic microbial identification system (Biolog, Hayward, CA, USA) for cultivation and automatic identification [33].

Screening of Virulence Genes
Eight virulence genes, namely, serine protease (ahp), flagella (fla), cytotoxic enterotoxin (act), elastase (ela), haemolysin (hly), heat-labile cytotonic enterotoxin (alt), lipase (lip) and aerolysin (aer), were screened using PCR. The primers used for amplification of the virulence genes are listed in Table 1. The amplification programs were similar to those used for 16S rRNA gene amplification reported previously, except that different annealing temperatures were used. The amplified products were analysed with 1% agarose gel electrophoresis and visualized using the ultraviolet light transilluminator (Bio-Rad).

Histopathological Analysis
Tissues of the liver, spleen, kidney and intestine were collected from diseased and healthy M. salmoides specimens. The tissues were fixed in 4% paraformaldehyde, dehydrated with an ethanol gradient series, embedded in paraffin and sectioned using a microtome (Thermo, Waltham, MA, USA), stained with haematoxylin-eosin (Solarbio, Beijing, China) and observed under an optical microscope (Olympus, Tokyo, Japan) [34].

Pathogenicity Assays
The pathogenicity assay was performed using healthy M. salmoides specimens, according to the methodology described in a previous study [37]. The median lethal dose (LD 50 ) was calculated using the improved Kohl's method [38]. The bacterial strain WH21406 was cultured in BHI at 28 • C for 20 h, harvested via centrifugation at 4 • C and 6000 rpm for 5 min and washed three times with sterile PBS. The bacteria were re-suspended in sterile PBS at a 10-fold ratio to 1.0 × 10 4 , 1.0 × 10 5 , 1.0 × 10 6 , 1.0 × 10 7 and 1.0 × 10 8 CFU mL −1 . The concentration of the bacterial suspension was determined using the plate colony counting method [38]. One hundred and eighty M. salmoides specimens were randomly divided into six groups (30 fish per group). The infected groups were intraperitoneally injected with 0.2 mL of each bacterial suspension (1.0 × 10 4 , 1.0 × 10 5 , 1.0 × 10 6 , 1.0 × 10 7 and 1.0 × 10 8 CFU mL −1 ). The control group was injected with 0.2 mL of sterile PBS. The experiment was repeated twice. The mortality of all groups was recorded daily for 14 days, and the dead fish were subjected to bacterial isolation and identification.

Clinical Symptoms
Diseased M. salmoides swam on the water surface and swam alone by the pond. Redness of the skin and mouth, ulcers and turgidity were observed ( Figure 1A). After dissection, liver yellowing and blood loss were observed. No ascites was found in the abdominal cavity, the kidney was swollen and the muscle was red and bleeding ( Figure 1B).

Pathogen Isolation and Characterization
Buff, translucent, circular and convex bacterial colonies were observed on the BHI agar plates after incubation for 24 h (Figure 2A). No discernible differences in color or form were detected among all colonies on the plates. Additionally, no viruses or parasites were detected in any of the sampled fish. The bacteria from the diseased fish were gramnegative and rod-shaped ( Figure 2B). The scanning electron microscope (Hitachi) also demonstrated that the bacteria were nearly rod-shaped (approximately 2.4 × 0.8 μm; Figure 2C).
The bacterial strain WH21406 was identified as Aeromonas caviae with the Biolog Automatic Microbial Identification System. According to the test report, the WH21406 matched the A. caviae reference strain, and comparison with the reference strain in the database yielded a satisfactory score ( Table 2).

Reagent
Result

Pathogen Isolation and Characterization
Buff, translucent, circular and convex bacterial colonies were observed on the BHI agar plates after incubation for 24 h (Figure 2A). No discernible differences in color or form were detected among all colonies on the plates. Additionally, no viruses or parasites were detected in any of the sampled fish. The bacteria from the diseased fish were gram-negative and rod-shaped ( Figure 2B). The scanning electron microscope (Hitachi) also demonstrated that the bacteria were nearly rod-shaped (approximately 2.4 × 0.8 µm; Figure 2C).

Pathogen Isolation and Characterization
Buff, translucent, circular and convex bacterial colonies were observed on the BHI agar plates after incubation for 24 h (Figure 2A). No discernible differences in color or form were detected among all colonies on the plates. Additionally, no viruses or parasites were detected in any of the sampled fish. The bacteria from the diseased fish were gramnegative and rod-shaped ( Figure 2B). The scanning electron microscope (Hitachi) also demonstrated that the bacteria were nearly rod-shaped (approximately 2.4 × 0.8 μm; Figure 2C).
The bacterial strain WH21406 was identified as Aeromonas caviae with the Biolog Automatic Microbial Identification System. According to the test report, the WH21406 matched the A. caviae reference strain, and comparison with the reference strain in the database yielded a satisfactory score (Table 2).

Reagent
Result * Reagent Result * A1 Negative Control N E1 Gelatin P A2 Dextrin P E2 Glycyl-L-Proline P A3 D-Maltose P E3 L-Alanine P A4 D-Trehalose P E4 L-Arginine P A5 D-Cellobiose P E5 L-Aspartic Acid P A6 Gentiobiose B E6 L-Glutamic Acid P A7 Sucrose P E7 L-Histidine P A8 D-Turanose B E8 L-Pyroglutamic Acid P A9 Stachyose B E9 L-Serine P The bacterial strain WH21406 was identified as Aeromonas caviae with the Biolog Automatic Microbial Identification System. According to the test report, the WH21406 matched the A. caviae reference strain, and comparison with the reference strain in the database yielded a satisfactory score ( Table 2).

Virulence Gene Assessment
According to the PCR profiles of the eight virulence genes, fla, aer, ela and hly were found in WH21406, and act, ahp, alt and lip genes were not detected (Figure 4).

Histopathological Observations
The tissues of the diseased fish showed obvious haemorrhaging and necrosis. In the diseased fish, a large number of inflammatory cells had infiltrated the liver ( Figure 5B, arrow), and the hepatocyte showed vacuolation ( Figure 5B, triangle). The splenic cells of the healthy fish were tightly arranged, but the splenic tissue of the diseased fish showed extensive necrocytosis ( Figure 5D, triangle) and hemosiderin deposits ( Figure 5D, asterisk). In the diseased fish, a large number of inflammatory cells had infiltrated the kidney ( Figure 5F, arrow), and necrosis was observed in the glomerulus (Figure 5F, triangle). In the intestine of the diseased fish, considerable damage and abnormal changes in the villi structure were detected ( Figure 5H, triangle). In the gills of the diseased fish, necrosis of the epithelia and swelling were observed ( Figure 5J, triangle).

Virulence Gene Assessment
According to the PCR profiles of the eight virulence genes, fla, aer, ela and hly were found in WH21406, and act, ahp, alt and lip genes were not detected (Figure 4).

Virulence Gene Assessment
According to the PCR profiles of the eight virulence genes, fla, aer, ela and hly were found in WH21406, and act, ahp, alt and lip genes were not detected (Figure 4).

Histopathological Observations
The tissues of the diseased fish showed obvious haemorrhaging and necrosis. In the diseased fish, a large number of inflammatory cells had infiltrated the liver ( Figure 5B, arrow), and the hepatocyte showed vacuolation ( Figure 5B, triangle). The splenic cells of the healthy fish were tightly arranged, but the splenic tissue of the diseased fish showed extensive necrocytosis ( Figure 5D, triangle) and hemosiderin deposits ( Figure 5D, asterisk). In the diseased fish, a large number of inflammatory cells had infiltrated the kidney ( Figure 5F, arrow), and necrosis was observed in the glomerulus (Figure 5F, triangle). In the intestine of the diseased fish, considerable damage and abnormal changes in the villi structure were detected ( Figure 5H, triangle). In the gills of the diseased fish, necrosis of the epithelia and swelling were observed ( Figure 5J, triangle).

Histopathological Observations
The tissues of the diseased fish showed obvious haemorrhaging and necrosis. In the diseased fish, a large number of inflammatory cells had infiltrated the liver ( Figure 5B, arrow), and the hepatocyte showed vacuolation ( Figure 5B, triangle). The splenic cells of the healthy fish were tightly arranged, but the splenic tissue of the diseased fish showed extensive necrocytosis ( Figure 5D, triangle) and hemosiderin deposits ( Figure 5D, asterisk). In the diseased fish, a large number of inflammatory cells had infiltrated the kidney ( Figure 5F, arrow), and necrosis was observed in the glomerulus ( Figure 5F, triangle). In the intestine of the diseased fish, considerable damage and abnormal changes in the villi structure were detected ( Figure 5H, triangle). In the gills of the diseased fish, necrosis of the epithelia and swelling were observed ( Figure 5J, triangle).

Antibiotic Susceptibility Test
The antibiotic susceptibility test demonstrated that strain WH21406 was highly sensitive to enrofloxacin, norfloxacin, streptomycin and amikacin and moderately sensitive to gentamicin (Table 3). Additionally, WH21406 was resistant to florfenicol, neomycin sulphate, compound sulfamethoxazole, doxycycline and tetracyclines.

Pathogenicity Assays
The cumulative survival rate of M. salmoides challenged with the isolate A. caviae WH21406 for 10 days is shown in Figure 6. The fish injected with A. caviae WH21406 died between the fourth and seventh day post-injection. The LD 50 of A. caviae WH21406 injected intraperitoneally was calculated to be 3.46 × 10 5 CFU mL −1 using the improved Kou's method. The symptoms of the artificially infected fish were consistent with those of the naturally infected fish, which showed ulcers and turgidity on the body surface and fin rot. No death or clinical symptoms were observed in the control group. Furthermore, A. caviae was re-isolated from the artificially infected fish. Mortality and symptoms were consistent in the repeated trials. These results indicated that the isolate A. caviae WH21406 was the pathogen.

Antibiotic Susceptibility Test
The antibiotic susceptibility test demonstrated that strain WH21406 was highly sensitive to enrofloxacin, norfloxacin, streptomycin and amikacin and moderately sensitive to gentamicin (Table 3). Additionally, WH21406 was resistant to florfenicol, neomycin sulphate, compound sulfamethoxazole, doxycycline and tetracyclines.

Pathogenicity Assays
The cumulative survival rate of M. salmoides challenged with the isolate A. caviae WH21406 for 10 days is shown in Figure 6. The fish injected with A. caviae WH21406 died between the fourth and seventh day post-injection. The LD50 of A. caviae WH21406 injected intraperitoneally was calculated to be 3.46 × 10 5 CFU mL −1 using the improved Kou's method. The symptoms of the artificially infected fish were consistent with those of the naturally infected fish, which showed ulcers and turgidity on the body surface and fin rot. No death or clinical symptoms were observed in the control group. Furthermore, A. caviae was re-isolated from the artificially infected fish. Mortality and symptoms were consistent in the repeated trials. These results indicated that the isolate A. caviae WH21406 was the pathogen.

Discussion
The genus Aeromonas contains many opportunistic pathogens that cause infections in many aquatic and terrestrial animals, including human beings [40][41][42]. A. caviae is a mesophilic species, and it is widely distributed in various aquatic ecosystems such as wastewater, aquaculture water and urban drinking water [18,43,44]. Recently, many studies revealed that the incidence of A. caviae infection in fish has increased, and the clinical symptoms are in accordance with the typical signs of Aeromonas spp. infection [23,44].
In this study, A. caviae WH21406 was isolated from diseased M. salmoides and identified using morphological characterization, biochemical identification and 16S rDNA sequence analysis. Additionally, the clinical signs of the artificially infected fish were similar to those of the naturally infected fish, and A. caviae was re-isolated from the artificially infected fish. These results indicated that A. caviae was the pathogen in the diseased fish. To the best of our knowledge, this is the first report of A. caviae infection in M. salmoides, and this strain caused a large number of deaths.
Virulence genes are good indicators of the pathogenicity of a microorganism [46]. We detected virulence genes fla, aer, ela and hly in A. caviae strain WH21406 (Figure 4). These virulence genes were extensively utilized to investigate the potential pathogenicity of Aeromonas spp. [47]. Flagella coded by fla are required for cellular propulsion. Motility is key for pathogenic bacteria that adhere to host cells and cause disease [25]. Aerolysin coded by aer is a pore-forming toxin that can damage epithelial cells in the intestine [48]. Elastase (ela), a secreted protein, can induce epithelial cell apoptosis. Haemolysins (hly) produce cytotoxic effects and lysis of erythrocytes, and they contribute to bacterial evasion from host inflammatory responses [49]. The virulence genes present in A. caviae WH21406 indicate that the synergistic effects conferred by combinations of these genes are key contributors to its high pathogenicity. And act, ahp, alt and lip genes were not detected in A. caviae WH21406. Mohamad [50] found that approximately 30 percent of A. caviae harbour ahp and lip genes. Some Aeromonas species establish in the gastrointestinal tract and can produce enteritis via elaboration of enterotoxigenic molecules such as cytotoxic enterotoxin (act) and cytotonic heat-labile enterotoxin (alt) [40]. No obvious enteritis symptoms occurred in the M. salmoides, which may be related to the absence of act and alt genes in A. caviae WH21406.
The histopathological analysis showed that the A. caviae isolate can cause obvious tissue damage and hemosiderin accumulation in the gill, liver, kidney, spleen and gut, which was similar to the damage found in Silurus meridionalis and Rhamdia quelen infected by A. caviae [23,51]. Cytopathic changes caused by several bacterial toxins may be an important cause of tissue damage. Previous studies indicated that haemolysin produced by A. caviae has a strong virulence and causes rupture and dissolution of red blood cells [23]. This may be the main reason for hemosiderin accumulation in the spleen and kidney.
Few studies were conducted regarding the antibiotic sensitivity of A. caviae. Antibiotic susceptibility testing for A. caviae WH21406 provided a reference for the treatment of A. caviae infection. The isolate WH21406 was highly sensitive to enrofloxacin, norfloxacin, streptomycin and amikacin and resistant to florfenicol, neomycin sulphate, compound sulfamethoxazole, doxycycline and tetracyclines ( Table 3). The A. caviae strain from Macrobrachium rosenbergii was highly sensitive to chloramphenicol, florfenicol, tetracycline and doxycycline and resistant to norfloxacin, erythromycin, streptomycin, compound sulfamethoxazole and rifampicin [52]. A. caviae strains from different fish exhibit different antibiotic sensitivity characterizations. Therefore, drug sensitivity testing is particularly important for the treatment of diseases in the production process.

Conclusions
In this study, pathogenic A. caviae WH21406 was isolated from diseased M. salmoides. The artificial infection test showed that A. caviae WH21406 is highly pathogenic to M. salmoides, and this isolate is highly sensitive to enrofloxacin, norfloxacin, streptomycin and amikacin. The results of this study provide a reference for the diagnosis and treatment of fish infection with A. caviae.