A composition for inactivating hepatoenterosporidial cysts in vitro and its application and method

Abstract

The present invention provides a composition for inactivating enterocystis in vitro, and its application and method, and relates to the field of aquaculture technology. The method for inactivating enterocystis in vitro of the present invention comprises: stimulating enterocystis with an aqueous solution having a pH value of 4.0-5.0, and then treating with a chlorine dioxide solution. The aqueous solution having a pH value of 4.0-5.0 stimulates enterocystis and can germinate it; the chlorine dioxide solution treatment can inactivate enterocystis and lose the ability to infect and cause disease. The method for inactivating enterocystis in vitro of the present invention is pollution-free to the environment, and in addition, it has certain advantages in terms of safety, effectiveness, cost-effectiveness, convenience, etc., and is highly targeted and effective.

Description

Technical Field

[0001] The invention belongs to the technical field of breeding, and in particular relates to a composition for inactivating hepatoenteroides in vitro and an application and method thereof. Background Art

[0002] The white shrimp (Litopenaeus vannamei) is one of the largest shrimp species currently farmed in my country. It is delicious, nutritious, and has high economic value, and is deeply loved by people. The white shrimp has a fast growth rate, a high reproduction number, and a high survival rate of offspring. It has become one of the important aquatic products in the Chinese market, and its market has broad development prospects. However, the shrimp farming industry has been plagued by the slow growth syndrome of shrimp. The growth rate of shrimp in the breeding pond with slow growth syndrome is significantly lower than that of normal shrimp. EHP is currently one of the main pathogens causing slow growth of shrimp.

[0003] Enterocytozoon hepatopenaei (EHP) is an obligate intracellular parasitic microsporidia with a chitinous spore wall and strong stress resistance. After infecting and parasitizing the host, it grows and develops by plundering or competing with the host for nutrients, causing malnutrition in the host, which in turn leads to slow or even stagnant growth, seriously affecting the production of adult shrimp in farmed shrimp and damaging the economic benefits of farmers. Every year, there are cases of failed farming due to EHP infection, and pond cleaning is required. The main transmission methods of EHP are vertical transmission and horizontal transmission. If the pond is not cleaned, there will be a risk of infection when farming again. Traditional disinfection methods mainly use CaO 2 This method is time-consuming, labor-intensive and costly. So far, there is no report on clear drugs that can control EHP infection. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a composition for inactivating hepatoenteroides cysts in vitro and its application and method, which can make hepatoenteroides lose its ability to infect and cause disease.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a composition for inactivating hepatoenteroides in vitro, comprising solutions with the following concentrations: a 50-100 mg / L chlorine dioxide solution and an aqueous solution with a pH value of 4.0-5.0.

[0007] Preferably, the pH value of the aqueous solution is adjusted by citric acid.

[0008] The invention also provides the use of the composition in inactivating hepatoenteroides in vitro.

[0009] The present invention also provides a method for inactivating hepatoenteroides cysts in vitro using the composition, comprising: treating a hepatoenteroides cyst suspension with the aqueous solution and then treating it with a chlorine dioxide solution.

[0010] Preferably, the method for preparing the hepatocystis suspension comprises: preparing the hepatopancreas of Penaeus vannamei infected with hepatocystis into a homogenate, performing density gradient centrifugation using Percoll with a volume percentage of 25%-35%, 55%-65% and 85-95%, and resuspending the bottom layer of precipitate with water to obtain the hepatocystis suspension.

[0011] Preferably, the volume of water used for resuspension is 90-110 μL.

[0012] Preferably, the volume ratio of the aqueous solution, the chlorine dioxide solution and the hepatocystis suspension is (1-3):(1-3):(1-3).

[0013] Preferably, the aqueous solution treatment time is 5-15 min.

[0014] Preferably, the treatment time of the chlorine dioxide solution is 10-20 hours.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides a composition for inactivating hepatoenteric cysts in vitro, and its application and method, comprising: stimulating hepatoenteric cysts with an aqueous solution having a pH value of 4.0-5.0, and then treating with a chlorine dioxide solution. The aqueous solution having a pH value of 4.0-5.0 stimulates the hepatoenteric cysts to cause them to germinate; the chlorine dioxide solution treatment can inactivate the hepatoenteric cysts and make them lose their ability to infect and cause disease. The method for inactivating hepatoenteric cysts in vitro of the present invention is environmentally friendly, and in addition has certain advantages in terms of safety, effectiveness, cost-effectiveness, convenience, etc., and is highly targeted and effective. . BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The density gradient centrifugation results of the separation and purification of EHP in Example 1 are shown;

[0018] Figure 2 The mixed solution of step 2 of Example 1 and Comparative Example 1 is microscopically examined; the left picture is Comparative Example 1, and the right picture is Example 1;

[0019] Figure 3 The effects of different concentrations of chlorine dioxide solution on EHP; the concentrations of chlorine dioxide solution in the upper left, upper middle, upper right, middle left, middle right, lower left and lower right pictures are 0, 0.5, 1, 5, 10, 50 and 100 mg / L respectively. DETAILED DESCRIPTION

[0020] The invention provides a composition for inactivating hepatoenteroides in vitro, comprising solutions with the following concentrations: a 50-100 mg / L chlorine dioxide solution and an aqueous solution with a pH value of 4.0-5.0.

[0021] In the present invention, there is no particular limitation on the agent for adjusting the pH value of the aqueous solution. As an implementation method, the pH value of the aqueous solution is adjusted by citric acid.

[0022] The water in the aqueous solution of the present invention is preferably ultrapure water.

[0023] The invention also provides the use of the composition in inactivating hepatoenteroides in vitro.

[0024] The present invention also provides a method for inactivating hepatoenteroides cysts in vitro using the above composition, comprising: stimulating a hepatoenteroides cyst suspension with the aqueous solution and then treating it with a chlorine dioxide solution.

[0025] One of the infection mechanisms of EHP is that spores recognize and bind to host cells, EHP spores germinate, polar filaments pop out and pierce the plasma membrane of host cells to become polar tubes, and sporoplasm is injected into host cells to complete infection, while inactivated EHP cannot pop out polar filaments, that is, it cannot inject sporoplasm into host cells to complete infection. Sporozoa exist in the form of dormant spores in vitro, and the spore wall is composed of chitin and spore wall protein, which can resist the adverse environment of the outside world. The spore wall structure of hepatoenteric spores makes it have a strong tolerance to environmental stress, and it can survive for a considerable period of time even if it leaves the host and enters the environment. Therefore, in the prior art, if EHP is not germinated, the pond is directly cleared with drugs, and EHP cannot be completely inactivated, resulting in repeated infection of EHP in white shrimp. The present invention first stimulates the hepatoenteric spores with a low pH aqueous solution to make it germinate, and then treats it with a chlorine dioxide solution to inactivate the hepatoenteric spores and lose the ability to infect and cause disease, which can significantly reduce the probability of white shrimp infected with EHP from the source.

[0026] The method for preparing the hepatocystis suspension of the present invention preferably comprises: preparing the hepatopancreas of Penaeus vannamei infected with hepatocystis into a homogenate, performing density gradient centrifugation with Percoll having a volume percentage of 25%-35%, 55%-65% and 85-95%, and resuspending the bottom layer of precipitation with water to obtain the hepatocystis suspension; the concentration of the Percoll is more preferably 30%, 60% and 90%; the volume of the water used for the resuspension is preferably 90-110 μL, more preferably 100 μL.

[0027] The volume ratio of the aqueous solution, the chlorine dioxide solution and the hepatocystis suspension of the present invention is preferably (1-3):(1-3):(1-3), and more preferably 2:2:2.

[0028] In the present invention, the treatment time of the aqueous solution is preferably 5-15 min, more preferably 10 min; the treatment time of the chlorine dioxide solution is preferably 10-20 h, more preferably 16 h.

[0029] The present invention has no special limitation on raw materials whose sources are not involved, and conventional commercial products in the field can be used.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1

[0032] 1. Separation and purification of EHP

[0033] (1) The hepatopancreas of 20 Penaeus vannamei infected with EHP was obtained, ground in a mortar and mortar, and filtered through a 0.22 μm filter membrane;

[0034] (2) Centrifuge at 2000 rpm for 10 min at room temperature, remove the supernatant and discard the precipitate;

[0035] (3) Centrifuge at 9000 rpm for 20 min at room temperature and discard the supernatant;

[0036] (4) After washing the precipitate with ultrapure water, centrifuge at 9000 rpm for 20 min at room temperature and discard the supernatant;

[0037] (5) Resuspend the obtained precipitate with 5 ml of ultrapure water, mix well and set aside;

[0038] (6) The EHP resuspension was centrifuged at 4500 rpm for 1 h using 30%, 60% and 90% Percoll (see results). Figure 1 );

[0039] (7) Take the bottom layer of EHP and place it in a 1.5 mL centrifuge tube, wash it with ultrapure water, centrifuge it at 9000 rpm for 10 min, and discard the supernatant;

[0040] (8) Resuspend the precipitate in 100 μL of ultrapure water for later use.

[0041] 2. Low pH ultrapure water treatment EHP

[0042] (1) Adjust the pH of ultrapure water to 4.5 with citric acid;

[0043] (2) Take 10 μL of EHP resuspension and 10 μL of pH 4.5 aqueous solution and incubate at room temperature for 10 min to obtain a mixed solution.

[0044] 3. Chlorine dioxide treatment of EHP

[0045] (1) Take the mixture from step 2 and add it to 1.5 mL of sterile EP;

[0046] (2) Add 10 μL of 50 mg / L chlorine dioxide solution to the above EP tube;

[0047] (3) Mix by gently pipetting;

[0048] (4) Incubate at room temperature for 16 h.

[0049] Example 2

[0050] The specific implementation is the same as that of Example 1, except that the concentration of the chlorine dioxide solution is 100 mg / L.

[0051] Comparative Example 1

[0052] The specific implementation is the same as that of Example 1, except that the pH value of the aqueous solution is 7.

[0053] Comparative Example 2

[0054] The specific implementation is the same as that of Example 1, except that the concentration of the chlorine dioxide solution is 0 mg / L.

[0055] Comparative Example 3

[0056] The specific implementation is the same as that of Example 1, except that the concentration of the chlorine dioxide solution is 0.5 mg / L.

[0057] Comparative Example 4

[0058] The specific implementation is the same as that of Example 1, except that the concentration of the chlorine dioxide solution is 1 mg / L.

[0059] Comparative Example 5

[0060] The specific implementation is the same as that of Example 1, except that the concentration of the chlorine dioxide solution is 5 mg / L.

[0061] Comparative Example 6

[0062] The specific implementation is the same as that of Example 1, except that the concentration of the chlorine dioxide solution is 10 mg / L.

[0063] Experimental Example 1

[0064] Take 2 μL of the mixed solution of Example 1 and Comparative Example 1, step 2, respectively, on a glass slide and observe under a microscope. The results are as follows: Figure 2 shown.

[0065] Depend on Figure 2It can be seen that after the ultrapure water treatment with pH 7, EHP has no polar filaments, while after the aqueous solution treatment with pH 4.5, most of the EHP has polar filaments (arrows in the right figure). Therefore, the present application adopts the aqueous solution treatment with pH 4.5 to stimulate the germination of EHP.

[0066] Experimental Example 2

[0067] Take 2 μL of the solution of Example 1-2 and Comparative Example 2-6 after incubation for 16 h on a glass slide, add 2 μL of fluorescent pink B staining solution (to facilitate observation of spores and polar filaments), and observe the ejection of EHP polar filaments under a microscope. The specific results are shown in Figure 3 .

[0068] Depend on Figure 3 It can be seen that a large number of polar filaments were ejected in the 0, 0.5, 1, 5 and 10 mg / L chlorine dioxide treatment groups (as shown by the arrows), which failed to inhibit the germination of EHP; while no polar filaments were ejected in the 50 and 100 mg / L treatment groups, which could effectively inhibit the germination of EHP. It is believed that these two concentrations of chlorine dioxide can effectively inactivate EHP.

[0069] In summary, an acidic aqueous solution with a pH of 4.5 can effectively stimulate the germination of EHP, and chlorine dioxide with a concentration of 50-100 mg / L can effectively inhibit the germination of EHP, even inactivate it.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a composition for inactivating hepatoenteric cysts in vitro in the preparation of a drug for inactivating hepatoenteric cysts in vitro, characterized in that: The composition includes solutions of the following concentrations: 50 or 100 mg / L chlorine dioxide solution and an aqueous solution with a pH value of 4.5; adjusting the pH value of the aqueous solution by citric acid; The water in the aqueous solution is ultrapure water; The application is to treat the hepatocystis suspension with the aqueous solution and then treat it with a chlorine dioxide solution; The preparation method of the hepatocystis suspension comprises: preparing the hepatopancreas of a white shrimp infected with hepatocystis into a homogenate, performing density gradient centrifugation using Percoll with a volume percentage of 30%, 60% and 90%, and resuspending the bottom layer of precipitation with water to obtain the hepatocystis suspension; The volume of water used for the resuspension was 100 μL; The volume ratio of the aqueous solution, the chlorine dioxide solution and the hepatocystis suspension is 2:2:2; The time for the aqueous solution treatment is 10 min; The treatment time of the chlorine dioxide solution is 16 hours.

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