Application of endomonas in serving as disease-resistant probiotics of shellfish

Abstract

The invention belongs to the technical field of aquaculture science, microbiology, immunology and bioengineering, and particularly relates to application of endophytic monad in serving as disease-resistant probiotics of shellfish. The added endophytic monad has a protection effect on the pathogen infected shellfish, the survival rate of the shellfish can be remarkably increased by adopting a seawater direct adding method of the two endophytic monad, namely a pre-adding method and a continuous adding method, and although the two tested adding methods have remarkable protection effects, the survival rate of the shellfish can be remarkably increased. However, compared with a continuous adding method, the pre-adding method is simpler, more convenient and more labor-saving in operation, and the pre-adding method is more recommended to be adopted in practical application; the principle for realizing protection is related to the growth inhibition of the endophytic monad on pathogenic bacteria, the reduction of host shellfish tissue damage and the improvement of the phagocytic activity of host shellfish blood cells. The invention provides a new strategy for improving disease resistance of shellfish and reducing disease loss of shellfish aiming at the current situations of difficulty in disease prevention and control and antibiotic abuse in current aquaculture, and can be applied to scenes such as healthy culture of shellfish and the like.

Description

Technical Field

[0001] The present invention belongs to the technical fields of aquaculture, microbiology, immunology, and bioengineering, and specifically relates to the application of an endophytic bacterium as a disease-resistant probiotic for shellfish. Background Art

[0002] Shellfish are one of the pillars of China's aquaculture industry. Diseases have always been an important problem faced by shellfish farming. In particular, the frequent occurrence of shellfish summer mortality causes serious economic losses. This disease is usually related to pathogenic bacteria such as Vibrio. Traditionally, antibiotics and other drugs have been used to combat pathogens in aquaculture. However, the overuse of antibiotics has led to the development of antibiotic-resistant pathogenic bacteria, posing serious risks to the ecology and human health. Therefore, finding drug-free methods to enhance the disease resistance of shellfish has become a crucial challenge faced by the aquaculture industry. More and more studies have shown that symbiotic microorganisms play a crucial role in maintaining the health of host animals, and further research is needed. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of an endophytic bacterium as a disease-resistant probiotic for shellfish.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] The application of an endophytic bacterium as a disease-resistant probiotic for shellfish.

[0006] The strain number of the endophytic bacterium is DSM 22380.

[0007] The application of the endophytic bacterium as a disease-resistant probiotic for aquaculture shellfish.

[0008] The application of the endophytic bacterium in resisting summer mortality disease as a disease-resistant probiotic for aquaculture shellfish.

[0009] The summer mortality disease resistant is the summer mortality of shellfish caused by pathogenic bacteria.

[0010] The pathogenic bacteria are opportunistic pathogenic bacteria such as Vibrio parahaemolyticus, Vibrio harveyi, or Vibrio alginolyticus.

[0011] A disease-resistant preparation for cultured shellfish, and the preparation contains an endophytic bacterium.

[0012] The preparation is a culture solution, culture, or resuspension of the endophytic bacterium.

[0013] The culture solution is obtained after the strain is activated. The precipitate separated after activation is the culture, and the culture is resuspended to obtain the resuspension.

[0014] Application of the described disease-resistant preparation, and application of the preparation as a disease-resistant probiotic for shellfish in aquaculture against summer death disease.

[0015] The preparation is added to the environment to be treated so that the concentration of the strains in the preparation in the environment reaches 1×10 3 -1×10 6 (optimally 1×10 5 ) CFU / ml.

[0016] The addition method of the preparation is pre-addition or continuous addition.

[0017] The cultured shellfish include bivalve shellfish such as Meretrix meretrix, Ruditapes philippinarum, Cyclina sinensis, Ostrea, Pecten, etc., and gastropod shellfish such as Haliotis discus hannai, etc.

[0018] Advantages of the present invention:

[0019] The method of the present invention can avoid the use of drugs and improve the disease resistance of shellfish in aquaculture by using a brand-new probiotic strategy. Previous studies on the probiotics of shellfish were basically intestinal probiotics. The present invention provides a brand-new method of using other symbiotic bacteria as disease-resistant probiotics, and this is also the first reported example of the disease resistance of Endomonas in shellfish. The present invention provides the specific details of using the addition of Endomonas to improve the disease resistance of shellfish. The actual operation is simple and easy, with remarkable effects and is convenient for application. Description of the drawings

[0020] Figure 1 Effect of adding Endomonas at different concentrations on the cumulative mortality of challenged Meretrix meretrix larvae.

[0021] Figure 2 Effect of adding Endomonas by two methods on the survival rate of challenged Meretrix meretrix.

[0022] Figure 3 Effect of adding Endomonas on the Vibrio load in challenged Meretrix meretrix.

[0023] Figure 4 Effect of adding Endomonas on the tissue damage of challenged Meretrix meretrix.

[0024] Figure 5 Effect of adding Endomonas on the phagocytic activity of hemocytes of challenged Meretrix meretrix.

[0025] Figure 6 Effect of Endomonas on the growth of Vibrio parahaemolyticus. Detailed implementation manners

[0026] The present invention will be further elaborated in the following examples, but the present invention is not limited thereto.

[0027] The present invention adds endophytic bacteria, which has a protective effect on shellfish infected by pathogens. Two methods of directly adding endophytic bacteria to seawater, namely the pre-adding method and the continuous adding method, can significantly improve the survival rate of shellfish. Although both of the tested adding methods have significant protective effects, considering that the pre-adding method is more convenient and labor-saving than the continuous adding method, the pre-adding method is more recommended for actual application; the principle of achieving protection is related to the growth inhibition of endophytic bacteria on pathogens, reducing host shellfish tissue damage, and improving the phagocytic activity of host shellfish blood cells. In view of the current difficulties in preventing and controlling aquaculture diseases and the abuse of antibiotics, the present invention provides a new strategy for improving the disease resistance of shellfish and reducing the loss of shellfish diseases, and can be applied to scenarios such as the healthy culture of shellfish.

[0028] In the following examples, the challenge was carried out by adding Vibrio parahaemolyticus to the water body. The strains used and the challenge method are detailed in the article doi:10.1016 / j.jip.2009.11.008. Briefly, after activation of Vibrio parahaemolyticus on a 2216E plate, it was inoculated into a 2216E liquid medium for propagation, the cells were obtained by centrifugation, and then added to the shellfish culture water body, with a final concentration of 5×10 7 CFU / ml.

[0029] The endophytic bacteria strain number used in the following examples is DSM 22380, which belongs to the strain cataloging system of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (abbreviated as DSMZ). After activation on a 2216E plate, it was propagated in a 2216E liquid medium until the logarithmic growth phase, and then added to the water environment to be detected.

[0030] Example 1. Determination of the optimal water addition concentration of endophytic bacteria

[0031] The optimal water addition concentration of endophytic bacteria was determined using Meretrix meretrix larvae. The specific method was as follows:

[0032] One blank control group (without adding Vibrio) and five Vibrio challenge groups (the final concentration of Vibrio parahaemolyticus in seawater: 1×10 6 CFU / ml) were set up respectively. The five Vibrio challenge groups were also added with 0, 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6Endophytic bacteria at the final concentration in seawater of CFU / ml. Each group was divided into three replicates (each replicate had approximately 80 clams) for recording the survival rate. During the experiment, Isochrysis galbana was fed to the clams daily in the conventional culture method, the seawater was renewed daily, and the corresponding bacteria were added daily according to the group to keep it consistent with the original dosage for 4 days.

[0033] The results showed (attached Figure 1 ): The addition of endophytic bacteria significantly reduced the mortality rate of Meretrix meretrix larvae, especially when the endophytic bacteria were added at the final concentration of 1×10 5 CFU / ml, showing the best protective effect. Therefore, 1×10 5 CFU / ml was determined as the optimal water body addition concentration of endophytic bacteria as a disease-resistant probiotic for shellfish.

[0034] Example 2. Determination of the effect of adding endophytic bacteria on the survival rate of diseased shellfish

[0035] Two addition methods were used to detect the protective effect of endophytic bacteria on adult Meretrix meretrix. The specific methods were as follows:

[0036] Adult Meretrix meretrix were randomly divided into four groups, with 80 in each group: 1 blank control group (without adding Vibrio) and 3 Vibrio challenge groups. The first Vibrio challenge group did not add endophytic bacteria (V group); the second group added endophytic bacteria 24 hours before Vibrio challenge (pre-addition: (E+V)-1 group); the third group added endophytic bacteria 24 hours before Vibrio challenge and during Vibrio challenge (continuous addition: (E+V)-2 group). Each group was divided into 4 replicates, among which 3 replicates were used to record the survival rate and 1 replicate was used for sampling for subsequent analysis. Vibrio parahaemolyticus was used for the immersion test, and the final concentration in seawater was 1×10 7 CFU / ml. The final addition concentration of endophytic bacteria was 1×10 5 CFU / ml. The seawater was renewed daily, and the corresponding bacteria were added daily according to the group to keep it consistent with the original dosage until the cumulative mortality rate of Meretrix meretrix reached 15%. The mortality rates of the 4 groups were recorded daily.

[0037] The results showed (attached Figure 2 ): The addition of endophytic bacteria significantly increased the survival rate of adult Meretrix meretrix after being infected with Vibrio. Both addition methods had obvious protective effects, and the protective effect of the pre-addition method was better than that of the continuous addition method. Therefore, adding endophytic bacteria in seawater can be used as a means to improve the disease resistance of shellfish.

[0038] To explain the principle of adding endophytic bacteria to improve the disease resistance of shellfish, the effect of adding endophytic bacteria on the Vibrio load in Meretrix meretrix was further detected. The specific methods were as follows:

[0039] The Meretrix meretrix tissues were homogenized in a homogenizer and spread on TCBS medium plates. After the plates were incubated at 28 °C for 24 hours, colony counting was performed. There were four clams in each group, and three replicates were set for each clam. The quantitative results were finally expressed as the number of colonies per gram of tissue.

[0040] The results showed (attached Figure 3 ): Compared with the control group V, the addition of Endozoicomonas significantly inhibited the increase in Vibrio load in the challenged Meretrix meretrix, which was related to the protective effect of Endozoicomonas addition on Meretrix meretrix.

[0041] The shellfish after the above treatments were detected

[0042] 1) To investigate the effect of Endozoicomonas addition on tissue damage in diseased shellfish:

[0043] The specific method was as follows:

[0044] The clams in the above different treatment groups were sectioned, stained with H&E method, and the histological characteristics differences of Meretrix meretrix in different treatment groups were evaluated by microscopic observation.

[0045] The results showed (attached Figure 4 ): Compared with the control group (C) without Vibrio infection, the Vibrio-infected group (V) showed wider gaps between gill filaments, karyolysis, and a large loss of epithelial cilia; the Endozoicomonas addition group, especially the pre-addition group ((E+V)-1), significantly alleviated these damages. This indicated that the addition of Endozoicomonas, especially using the pre-addition method, effectively reduced the tissue damage caused by Vibrio infection, which was related to the protective effect of Endozoicomonas addition on Meretrix meretrix.

[0046] 2) The effect of Endozoicomonas addition on the hemocyte composition and phagocytic activity of diseased shellfish

[0047] The specific method was as follows:

[0048] For the clams in the above different treatment groups, their hemolymph was collected, centrifuged at 600 rpm for 10 minutes at 4 °C, and the cell pellet was resuspended to a final concentration of about 1×10 6 cells / ml. The prepared hemocyte suspension was incubated with fluorescent microspheres (Polysciences, USA) at a concentration of 1×10 7 particles / ml in the dark at 37 °C for 30 minutes. Subsequently, the hemocytes were washed three times to remove the unbound microspheres. The phagocytic activity of hemocytes in different treatment groups was evaluated and compared using a BD FACSAria flow cytometer based on whether they ingested the fluorescent microspheres.

[0049] The results showed (attached Figure 5):Flow cytometry analysis showed that the addition of Endozoicomonas increased the phagocytic activity of hemocytes during pathogen challenge. The (E+V)-1 group showed significantly enhanced phagocytic activity at 3 dpi, while the (E+V)-2 group showed significantly enhanced phagocytic activity at 10 dpi. This result was consistent with the increase in semi-granular cells. The results indicated that the addition of Endozoicomonas enhanced the phagocytic activity of hemocytes by increasing the proportion of semi-granular cells, thereby improving the disease resistance of Meretrix meretrix, which was related to the protective effect of Endozoicomonas addition on Meretrix meretrix.

[0050] Example 3. Detection of the antibacterial activity of Endozoicomonas against Vibrio

[0051] One control group (V group: only Vibrio parahaemolyticus added) and one experimental group (E+V group: Vibrio parahaemolyticus and Endozoicomonas added) were set up. Each group contained 3 replicated glass tubes, and each tube had 8 ml of sterilized 2216E liquid medium. All test tubes were cultured on a shaker at 28 °C at a speed of 150 rpm. During this period, 200 μL was removed every 2 hours. Then, 20 μL was spread on a TCBS medium plate. The plates were incubated at 28 °C for 24 hours and then colony counts were performed. The addition amount of Vibrio parahaemolyticus in each test tube was 1×10 3 CFU / ml; the addition amount of Endozoicomonas was 1×10 5 CFU / ml.

[0052] The results showed (see Figure 6 ): Compared with the control group, the Vibrio concentration in the group with Endozoicomonas addition was significantly reduced, indicating that Endozoicomonas had an inhibitory effect on the growth of Vibrio parahaemolyticus, which was related to the protective effect of Endozoicomonas on Meretrix meretrix.

[0053] Based on the above examples, we determined that the addition of Endozoicomonas could significantly improve the disease resistance of shellfish. The principle of achieving protection was related to the growth inhibition of Endozoicomonas on pathogenic bacteria, the reduction of host shellfish tissue damage, and the improvement of host shellfish hemocyte phagocytic activity. When applied, the optimal water addition concentration of Endozoicomonas as a disease-resistant probiotic for shellfish was 1×10 5 CFU / ml. Although both of the tested addition methods had significant protective effects, considering that the pre-addition method was more convenient and labor-saving than the continuous addition method, the pre-addition method was more recommended for actual application.

Claims

1. Application of an endophytic monospore as a probiotic for shellfish disease resistance.

2. The use according to claim 1, characterized in that: The endophytic omonas is used as a disease-resistant probiotic for aquaculture shellfish.

3. The use according to claim 2, characterized in that: The endophytic monad is used as a disease-resistant probiotic for aquaculture shellfish to fight summer mortality.

4. The use according to claim 3, characterized in that: The anti-summer mortality disease is the summer mortality of shellfish caused by pathogenic bacteria.

5. The use according to claim 4, characterized in that: Said anti-Vibrio parahaemolyticus, Vibrio harveyi or Vibrio alginolyticus.

6. A disease-resistant preparation for cultured shellfish, characterized in that: The preparation contains endogenous spores.

7. The anti-disease preparation according to claim 6, characterized in that: The preparation is a culture solution, a culture or a resuspension of the endophytic omonas.

8. Use of the anti-disease preparation according to claim 6, characterized in that: The preparation is used as disease-resistant probiotics for aquaculture shellfish to resist summer mortality.

9. The use of the anti-disease preparation according to claim 8, characterized in that: The preparation is added to the environment to be treated so that the concentration of the strain in the preparation in the environment reaches 1×10 3 -1×10 6 CFU / ml.

10. The use of the anti-disease preparation according to claim 9, characterized in that: The preparation is added in a pre-addition or continuous addition manner.

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