Study on Aeromonas hydrophila disease in European eel

In recent years, large-scale intensive farming of European eel has been increasing in China. However, with the expansion of farming scale and the continuous increase in farming density, various problems affecting the survival rate of European eel have also been increasing, and the fulminant infectious disease of European eel caused by Aeromonas hydrophila as the pathogen is one of the prominent problems. This paper conducts a series of preliminary studies on the physiological and biochemical characteristics, pathogenic factors, and pathogenic conditions of Aeromonas hydrophila, aiming to provide a strong theoretical basis for the prevention and control of fulminant infectious diseases in European eel, and to better promote the development of the European eel farming industry toward a stronger, faster, and healthier direction.

1. Materials and Methods

1. Isolation of pathogenic bacteria

Ordinary protein agar medium was used, with the composition of tryptone 10 g, yeast extract 10 g, sodium chloride 5 g, agar 18-20 g, pH 7.2-7.4, sterilized at 15 psi for 20 minutes and set aside.

Eels with typical symptoms were taken, and the ulcerated surface layer was cut open with a sterile scalpel. A small amount of mucus from deep in the lesion was quickly picked up with an inoculation loop and streaked onto agar plates for isolation. After routine culture at 28°C for 24 hours, some colonies with basically consistent morphology appeared. Single colonies were picked for pure culture, then transferred to slants and set aside.

2. Pathogenicity experiment

The experimental strains were inoculated onto slants and cultured at 28°C for about 24 hours. 5-10 mL of sterile physiological saline was added to each slant to prepare a thick bacterial suspension. 1 mL of the bacterial suspension was taken, and the bacterial count was determined by the viable plate count method. 0.1 mL of the bacterial suspension was drawn and injected into the abdominal cavity of healthy eels (which had been observed in aquariums for 5 days before injection). A control group was also set up, and the fish were placed in aquariums at 24-28°C to observe symptoms.

3. Morphological observation and physiological and biochemical experiments of pathogenic bacteria

Strains Ah3, Ah26, and Ah38, which had strong pathogenicity, were cultured at 28°C for 24 hours, then subjected to Gram staining and flagella staining to observe their morphology and conduct other physiological and biochemical experiments.

4. Serotype study

1) White rabbits (purchased from the market) were used to prepare antiserum. Healthy eels (provided by an eel farm) with a size of about 40-50 g were used for challenge, and were observed for 5-7 days before the experiment to confirm they were healthy and disease-free.

2) Strains Ah3, Ah26, Ah38, etc., which had strong pathogenicity, were cultured on nutrient agar for 24 hours respectively. The bacterial lawn was scraped off, and the bacterial cells were washed three times with sterile physiological saline. The cells were inactivated with 0.35% formalin for 25 hours to prepare whole-cell antigen (WC antigen), and stored in a refrigerator at 4°C for later use;

3) 0.1 mL of 0.35% formalin-inactivated cells (concentration 1×10E9 cell/mL) was mixed evenly with an equal amount of Freund's complete adjuvant, and subcutaneously injected into white rabbits at multiple points for immunization. Thereafter, booster immunization was performed via the ear vein every other week, with the antigen dose gradually increased each time. After satisfactory titer was obtained, blood was collected from the carotid artery to obtain serum.

4) Tube agglutination method: 0.25 mL of rabbit antiserum (1:60 dilution) was diluted, and 0.25 mL of antigen at a concentration of 15 R/mL was added, shaken well, kept at 4°C for 12 hours, and the agglutination reaction was observed.

5. Determination of virulence (LD50) of Aeromonas hydrophila

Ah strains were inoculated into nutrient broth and cultured at 28°C for 24 hours. The bacterial concentration was measured, and the bacterial cells were serially diluted 10-fold with sterile physiological saline. Healthy eels were injected intraperitoneally. The experimental water temperature was 25°C. The number of dead fish and the injected bacterial dose were recorded, and the LD50 was calculated by the Reed-Muench method.

6. Determination of hemolytic activity

Pure isolates of Ah strains were inoculated onto 10% rabbit blood plates and cultured overnight at 28°C. The ratio of the hemolytic zone diameter to the colony diameter was used to indicate hemolytic activity.

7. Exploratory determination of pathogenic conditions

Strain Ah38, which had strong pathogenicity, was used as the challenge strain. The water environment in the aquarium was adjusted so that the bacterial content was approximately 5-104 cells/mL. Under different conditions, healthy eels were immersed in the water to observe the conditional pathogenicity of Aeromonas hydrophila.

2. Experimental Results

1. In this experiment, a total of 63 strains were isolated, among which 8 strains were pathogenic through artificial infection. Among these 8 strains, 3 strains had extremely strong pathogenicity.

2. Morphological observation and physiological and biochemical characteristics of pathogenic bacteria.

The results of this experiment showed that the morphology, cultural characteristics, and physiological and biochemical reactions of strains Ah3, Ah6, Ah7, An26, Ah31, Ah38, Ah51, Ah53, etc., isolated from diseased fish were basically consistent. Strain Ah38 is briefly described as follows as a representative:

On nutrient agar plates, cultured at 28°C for 18-24 hours, the colonies were round, moist, with a smooth surface, low convex, with neat edges, semi-transparent, milky white, about 1-1.5 mm in diameter, with an off odor. In broth medium, cultured at 28°C for 18-24 hours, the culture liquid was uniformly turbid, with a mist-like film on the surface that dispersed upon shaking. Under a high-power microscope, it appeared as straight rods with rounded ends, single or in pairs, motile, without spores, without capsules, Gram-negative. Electron microscopy showed a single polar flagellum.

Based on these cultural characteristics, we confirmed that the isolated strains belong to Aeromonas hydrophila. They are the main pathogens causing fulminant infectious diseases in European eels, which has been widely recognized by most scholars.

3. We prepared rabbit antiserum against strains Ah3, Ah26, and Ah38, which had strong pathogenicity in the pathogenicity experiment, and conducted tube agglutination tests with the other 5 Ah strains. The results showed that Ah3 and Ah38 belonged to the same serotype, Ah26 belonged to another serotype, and the remaining 5 strains belonged to these two serotypes. The reaction specificity was strong, and no strain was found that could react with both serotypes simultaneously.

4. The results of the LD50 determination experiment of Aeromonas hydrophila showed that all eight Ah strains had strong virulence to healthy eels. The strongest strain, Ah38, had an LD50 lower than 104 CFU. At the same time, strains with strong virulence had a ratio of hemolytic zone diameter to colony diameter greater than 2, indicating that they had strong hemolytic activity. Other strains with lower LD50 also had lower hemolytic activity. This experimental result suggests that the virulence of Aeromonas hydrophila has a certain linear relationship with its hemolytic activity. Strains of different serotypes have inconsistent hemolytic ability. In our experiment, the serotype represented by strain Ah38 had stronger pathogenicity. This provides a new idea for us to seek effective biotechnology for the prevention and treatment of Aeromonas hydrophila infection in the future.

5. The results of the conditional pathogenicity experiment of Aeromonas hydrophila showed that when the water body contains a certain amount of Aeromonas hydrophila, if the fish is healthy and the water quality is good, then the probability of the fish being infected with the disease is very small; but when the fish itself is damaged to a certain extent, for example, gill filaments are damaged or the epidermis is damaged, especially when the gill filaments are damaged, the fish is very easily infected by bacteria and develops disease; and when the fish is injured and the water environment is also poor, the death rate and mortality of the fish are highest, as is self-evident, just as our experiment showed.

3. Discussion

As an inherent bacterial flora in water, Aeromonas hydrophila has become one of the main pathogens in freshwater fish farming diseases due to its characteristics of wide host range and strong infectivity. The highly pathogenic strains Ah3, Ah26, and Ah38 isolated in the experiment respectively represent two different serotypes, and they constitute the main pathogens causing fulminant infectious diseases of freshwater fish in the Jiangsu, Zhejiang, and Shanghai area. At the same time, virulence experiments and hemolytic activity experiments also showed that strains with strong virulence also have strong hemolytic activity. This seems to be the reason why Aeromonas hydrophila causes large-scale ulcerative bleeding in fish, and this result is basically consistent with the results reported in some literature. These scholars also believe that Aeromonas hydrophila can cause bleeding, hemolysis, and death in fish, and that its production of hemolysin, botulinum toxin, and cytotoxin may be related to pathogenic factors. This experiment also showed that different strains have different toxicity intensities. We speculate that some strains have only one virulence factor, while others have multiple, which is consistent with some views of certain scholars.

Regarding the infection route of Aeromonas hydrophila, we preliminarily speculate that the gills are the main route, and after infection, the liver is the main site of proliferation, which then spreads diffusely throughout the body. In this experiment, our pathogenic strains were mainly isolated from the liver, and after re-infection, bacteria appeared earliest in the liver, indicating that Aeromonas hydrophila infection also has a certain tropism.

Ammonia nitrogen and nitrite cannot provide nutrients for the growth of Aeromonas hydrophila, but excessively high content is an important environmental factor leading to fish disease. The reason is that nitrite nitrogen can destroy hemoglobin in the body's blood, causing red blood cells to lose oxygen-carrying capacity, while ammonia nitrogen not only directly causes fish poisoning, but also continuously decomposes to produce nitrite nitrogen, thereby reducing the fish's disease resistance and making it susceptible to bacterial infection and illness. In our experiment, when the ammonia nitrogen and nitrite nitrogen values were only twice the normal values, the mortality rate of eels was significantly higher than that of the control group, which also illustrates this point. Therefore, we preliminarily believe that Aeromonas hydrophila, as the main pathogen causing fulminant infectious diseases in freshwater fish, is a conditional pathogen, and its infectivity is related to the health status of the fish and the water environment. In recent years, a large number of emerging literature reports that the drug resistance range and intensity of Aeromonas hydrophila are continuously expanding and strengthening, and drug residues have posed a serious threat to human health, especially that of children. In practical work, we should shift our focus to controlling the @#@233@#@ environment, protecting water quality, and reducing bacterial infection routes. This is the fundamental way to prevent and treat fish diseases.