A STUDY ON HYPOPHILA DISEASE OF EUPHILA EELLUS

In recent years, the large-scale intensive culture of European eel has been increasing in China. However, with the expansion of culture scale and the continuous improvement of culture density, various problems affecting the survival rate of European eel are also increasing, and the fulminant infectious disease of European eel caused by Aeromonas hydrophila is one of the prominent problems. In this paper, a series of preliminary studies were made on the physiological and biochemical characteristics, pathogenic factors and pathogenic conditions of Aeromonas hydrophila, in order to provide a strong theoretical basis for the prevention and treatment of fulminant infectious diseases of European eel. To better promote the European eel breeding industry to a stronger, faster and healthier direction.

I. Materials and methods

1. Isolation of pathogenic bacteria

The common protein Agar medium was used, which was composed of tryptone 10g, yeast extract 10g, sodium chloride 5g, Agar 18g, 20g, pH7.2g, 7.4g and sterilized for 20 minutes.

The eel suffering from typical disease was cut open with aseptic scalpel and a little deep mucus was quickly picked up by inoculation ring and separated by Agar plate marking. After 28 ℃ and 24 hours of routine culture, some colonies with the same morphology appeared. Pick out a single colony, carry out pure culture, and then transfer the bevel for backup.

two。 Pathogenicity experiment

The experimental bacteria were connected to the inclined plane and cultured for about 24 hours at 28 ℃. Aseptic saline 5~10mL was added to each slope to make a thick bacterial suspension. The suspension of 1mL bacteria was taken and the number of bacteria was determined by the method of living bacteria plate counting. The suspension of 0.1mL bacteria was absorbed and injected into the abdominal cavity of healthy eel (observed in the aquarium for 5 days before injection). At the same time, the symptoms were observed in the aquarium of 24-28 ℃.

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

Strains Ah3, Ah26 and Ah38 with strong pathogenicity were cultured for 24 hours under the condition of 28 ℃. Gram staining and flagella staining were performed to observe their morphology and other physiological and biochemical experiments.

4. Serotype study

The main results are as follows: 1) antiserum was prepared by using small white rabbits (purchased from the market). The healthy eel (provided by the eel farm) was used to attack the virus, the specification was about 40g and 50g, and it was confirmed to be healthy and disease-free by observation for 5 to 7 days before the experiment.

2) Ah3, Ah26, Ah38 and other strains with strong pathogenicity were cultured on nutrient Agar for 24 hours, the moss was scraped, the bacteria were washed with sterile normal saline for three times, and the bacteria were inactivated with 0.35% formalin for 25 hours. The whole bacterial antigen (WC antigen) was prepared and stored in 4 ℃ refrigerator.

3) the rabbits were evenly mixed with 0.35% formalin inactivated 0.1mL (concentration 1 × 10E9cell/mL) and the same amount of Freund's complete adjuvant, and the rabbits were immunized subcutaneously at multiple points. After that, the rabbits were immunized by ear vein every other week, and the antigen dose was increased step by step. After the satisfactory titer was obtained, the blood was released from the carotid artery to take serum.

4) Test tube coacervation method: 0.25mL was diluted with immune antiserum (1 ∶ 60), 0.25mL concentration was added to 15R/mL antigen, shake well, 4 ℃, 12 hours, and the agglutination reaction was observed.

5. Virulence (LD50) determination of Aeromonas hydrophila

Ah strain was inoculated with nutritious broth and cultured for 24 hours at 28 ℃. The bacteria concentration was measured. Healthy eel was injected intraperitoneally with 10 times of sterile normal saline. The experimental water temperature was 25 ℃. The number of dead fish and the amount of bacteria injected were recorded, and LD50 was calculated according to Reed-Muench method.

6. Determination of hemolytic activity

The pure isolate of Ah strain was inoculated on 10% rabbit blood plate and cultured overnight at 28 ℃. The hemolytic activity was shown by the ratio of hemolytic circle diameter to colony.

7. Exploratory determination of pathogenic conditions

The pathogenic strain Ah38 was used as the attacking strain to adjust the water environment in the aquarium so that its bacterial content was about 5~104cells/mL. Under different conditions, the healthy eel was bathed in it to observe the conditional pathogenicity of Aeromonas hydrophila.

Second, the experimental results

1. In this experiment, a total of 63 strains were isolated, of which 8 strains had pathogenicity by artificial infection, and among these 8 strains, 3 strains had strong pathogenicity.

two。 Morphological observation and physiological and biochemical characteristics of pathogenic bacteria.

The results showed that the morphology, culture characteristics and physiological and biochemical reactions of Ah3, Ah6, Ah7, An26, Ah31, Ah38, Ah51, Ah53 and other strains isolated from diseased fish were basically the same. The representative of strain Ah38 is as follows:

On the nutrient Agar plate, the colony was round, moist, smooth, low convex, neat edge, translucent, milky white, about 1~1.5mm in diameter and smelly at 28 ℃ for 24 hours. In the broth medium, the culture medium was uniform and turbid at 28 ℃ for 24 hours, and the surface was foggy and dispersed as soon as it was shaken. Under high-power microscope, there were round straight bacilli at both ends, single or paired, capable of movement, no spores, no capsule, negative Gram staining, and solitary flagella under electron microscope.

According to these culture characteristics, we confirmed that the isolated strains belong to Aeromonas hydrophila (Aeromonushydrophila), which is the main pathogen of the outbreak of European eel infectious diseases, which has been recognized by most scholars.

3. We prepared rabbit antiserum from strains Ah3, Ah26 and Ah38 with strong pathogenicity in pathogenicity test, and carried out test tube agglutination test with other 5 strains of Ah. The results showed that Ah3 and Ah38 belonged to the same serotype, Ah26 belonged to another serotype, and the other 5 strains belonged to these two serotypes with strong reaction specificity, and no strains capable of reacting with the two serotypes were found.

4. The results of virulence test (LD50) of Aeromonas hydrophila showed that all the eight strains of Ah had strong virulence to healthy eel, and the strongest strain Ah38 had a lower LD50 than 104CFU. At the same time, the ratio of hemolytic circle diameter to colony diameter of the virulent strain was more than 2, indicating that it had strong hemolytic activity, and other strains with lower LD50 had lower hemolytic activity. The result of this experiment reveals that there is a certain linear relationship between the virulence of Aeromonas hydrophila and its hemolytic activity, and the hemolytic ability of different serotypes is different. In our experiment, the serotype represented by strain Ah38 has stronger pathogenicity, which provides a new idea for us to find effective biotechnology for the prevention and treatment of Aeromonas hydrophila infection in the future.

5. The experimental results of conditional pathogenicity of Aeromonas hydrophila show that when there is a certain amount of Aeromonas hydrophila in the water, if the healthy water quality of the fish is good, then the probability of infection of the fish is very small, but when the fish itself is damaged to a certain extent, for example, when the mumps or epidermis are damaged, especially when the Gill filaments are damaged, the fish are very vulnerable to bacterial infection. When the fish is injured and the water environment is poor, the highest death rate and mortality rate of fish is self-evident, as our experiments show.

III. Discussion

As an inherent flora in water, Aeromonas hydrophila has become one of the main pathogens of freshwater fish culture diseases because of its wide host range and strong infectivity. The highly virulent strains Ah3 and Ah26;Ah38 isolated in the experiment represent two different serotypes respectively, and they constitute the main pathogen causing fulminant infectious diseases of freshwater fish in Jiangsu, Zhejiang and Shanghai areas. at the same time, virulence and hemolytic activity tests also show that the virulent strains also have strong hemolytic activity. This seems to be the cause of large area ulcer bleeding in fish caused by Aeromonas hydrophila, which is basically consistent with the results reported in some literatures. These scholars also believe that Aeromonas hydrophila can cause bleeding, hemolysis and death in fish, and its production may be related to hemolysin, botulinum toxin and cytotoxin. This experiment also shows that the toxicity of different strains is different. We speculate that some strains have only one toxic factor, while others have multiple toxic factors, which is consistent with some views of some scholars.

With regard to the infection pathway of Aeromonas hydrophila, we speculate that the gills are the main pathway, and the liver is the main proliferation site after infection, and then spread to the whole body diffusely. In this experiment, our pathogenic strains were mainly isolated from the liver, and the earliest bacteria appeared in the liver after reinfection, indicating that Aeromonas hydrophila infection still has a certain affinity.

Ammonia nitrite can not provide nutrition for the growth of Aeromonas hydrophila, but high content is an important environmental factor leading to the disease of fish. The reason is that nitrite nitrogen can destroy the hemoglobin in the blood of the body and make red blood cells lose their ability to carry oxygen, while ammonia nitrogen not only directly causes fish poisoning, but also continues to decompose to produce nitrite nitrogen, thus reducing the disease resistance of fish. Vulnerable to bacterial infection and disease. In our experiment, the values of ammonia nitrogen and nitrite nitrogen were only twice the normal value, and the mortality rate of eel was significantly higher than that of the control group. Therefore, we believe that Aeromonas hydrophila, as the main pathogen causing fulminant infectious diseases of freshwater fish, belongs to conditional pathogens, and its infectivity is related to the health status of fish and water environment. In recent years, a large number of literatures have reported that the scope and intensity of drug resistance of Aeromonas hydrophila are constantly expanding and strengthening, and drug residues have posed a serious threat to the health of human beings, especially children. In practice, we should focus on controlling the environment. Protecting water quality and reducing the ways of bacterial infection are fundamental to the prevention and control of fish diseases.