Inactivated vaccine for epizootic haematopoietic necrosis disease in carassius auratus and preparation method of inactivated vaccine for epizootic haematopoietic necrosis disease in carassius auratus

A technology of hematopoietic organ necrosis and inactivated vaccine is applied in the field of crucian carp hematopoietic necrosis disease inactivated vaccine and its preparation field, which can solve the problems of economic loss of crucian carp breeding industry, threat to the healthy development of the industry, strong virus infectivity, etc., and achieve immune protection. Good effect, low production cost, good safety performance

Pending Publication Date: 2020-03-20
ZHEJIANG INST OF FRESH WATER FISHERIES +1
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Problems solved by technology

The virus is highly contagious and has a high fatality rate, which has caused major economic losses t

Method used

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  • Inactivated vaccine for epizootic haematopoietic necrosis disease in carassius auratus and preparation method of inactivated vaccine for epizootic haematopoietic necrosis disease in carassius auratus
  • Inactivated vaccine for epizootic haematopoietic necrosis disease in carassius auratus and preparation method of inactivated vaccine for epizootic haematopoietic necrosis disease in carassius auratus
  • Inactivated vaccine for epizootic haematopoietic necrosis disease in carassius auratus and preparation method of inactivated vaccine for epizootic haematopoietic necrosis disease in carassius auratus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The present embodiment provides a preparation method of crucian carp hematopoietic necrosis inactivated vaccine, comprising the following steps:

[0024] Step 1, CSC cell culture: take out the spinal cord tissue of heterogeneous gibel crucian carp under aseptic conditions, and process it into a size of 30-60mm 3 Put the tissue block into a culture dish containing L15 culture medium; put the tissue block evenly into a T25 cell culture bottle with the side of the tissue block facing up, add 3ml of L15 culture solution to the culture bottle, overnight, slowly Turn the culture bottle sideways to soak the tissue block in the culture medium, and then turn the side with the tissue block upwards, operate once from time to time until CSC cells grow on the edge of the tissue block, and culture the cell culture bottle upright, every 2 to 3 Replace the culture medium once a day; take 1 bottle of CSC covered with a single layer, discard the old medium in a sterile ultra-clean bench,...

Embodiment 2

[0033] Determination of CyHV-2 inactivation conditions and safety test.

[0034] Obtain the amplified virus stock solution according to the method of Example 1, divide it into 3 equal parts, add BEI to the final concentrations of BEI respectively 5mmol / L, 8mmol / L, and 10mmol / L, and inactivate it in a constant temperature shaker at 37°C at 120r / min After 24h, 48h, and 72h, use the same concentration of sodium thiosulfate solution to terminate the inactivation and take samples. Take the prepared vaccine and inoculate CSC cells according to the above virus propagation method, and set up a negative control at the same time, observe for 7 to 10 days, if the cytopathic effect appears, it indicates that the virus inactivation is not complete; if no cytopathic effect is seen, then blindly pass 2 Second, if cytopathy occurs in the blind transmission, it indicates that the virus inactivation is still incomplete. If no cytopathy occurs in the two blind transmissions, it indicates that th...

Embodiment 3

[0041] Safety Test of Inactivated Vaccines

[0042] Sterility test: Take the vaccine prepared in Example 1, inoculate the brain infusion bacterial medium (BHI) plate, apply the streak method on the plate, and cultivate it at 30°C for 15 days. If there is bacterial colony growth, it indicates that the vaccine has bacterial contamination. ; if no colonies grow, the vaccine is sterile.

[0043] Fish body safety test: Take the vaccine prepared above and inject 30 healthy crucian carp of about 300g, the injection dose is 0.2-0.4ml / tail, and the negative control is injected with the same dose of normal saline. After feeding for 15 to 30 days, if the vaccine group died or had clinical symptoms, but the negative control group did not show clinical symptoms or died, it indicated that the vaccine was unsafe; if neither the vaccine group nor the negative control group showed clinical symptoms or died, it indicated that the vaccine Safety.

[0044] Stress test and food intake effect aft...

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Abstract

The invention provides an inactivated vaccine for an epizootic haematopoietic necrosis disease in carassius auratus and a preparation method of the inactivated vaccine for the epizootic haematopoieticnecrosis disease in the carassius auratus. The vaccine comprises spinal cord tissue cell lines of carassius auratus gibelio and cyprinidherpesvirus II, and a preservation number of the spinal cord tissue cell lines of the carassius auratus gibelio is CCTCC NO: C2018211. The preparation method comprises the following steps: performing CyHV-2 virus amplification on the cultured spinal cord tissue cell lines of the carassius auratus gibelio to obtain CyHV-2 virus solutions; and performing inactivation treatment on the CyHV-2 virus solutions. The vaccine prepared by adopting the method disclosedby the invention has a good immunoprotection effect, can be applied to preventive immunity for the epizootic haematopoietic necrosis disease in the carassius auratus, and can increase survival ratiosof raised carassius auratus and the raising efficiency of the carassius auratus.

Description

technical field [0001] The invention belongs to the technical field of veterinary biomedicine, and in particular relates to an inactivated vaccine for crucian carp hematopoietic organ necrosis and a preparation method thereof. Background technique [0002] Crucian carp hematopoietic organ necrosis (commonly known as crucian gill hemorrhagic disease) is caused by Cyprinidherpesvirus Ⅱ (Cyprinidherpesvirus Ⅱ, CyHV-2) infection, and the other two herpesviruses CyHV-1 (Carp pox) and CyHV- 3 (Koi herpesvirus, KHV) belong to the Alloherpesviridae family, Cyprinivirus genus. CyHV-2 was first reported in 1995, and caused huge economic losses to goldfish cultured in western Japan from 1992 to 1993, and the mortality rate of sick goldfish was as high as 100%. International trade in ornamental fish has largely contributed to the global spread of the disease. In 2011, Hungary reported that CyHV-2 infection was also found in cultured gibel carp. Since 2009, crucian carp hematopoietic ...

Claims

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Application Information

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IPC IPC(8): A61K39/245A61P31/22C12N5/079C12N7/00C12N7/02
CPCA61K39/12A61P31/22C12N5/0618C12N7/00A61K2039/552A61K2039/5252C12N2710/16034C12N2710/16051
Inventor 沈锦玉曹铮潘晓艺夏焱春姚嘉赟蔺凌云尹文林刘忆瀚陆裕肖邢刚岳丰雄黄杰
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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