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A method for weakening the virulence of streptococcus agalactiae derived from tilapia

A technology of streptococcus lactis and tilapia, applied in biochemical equipment and methods, methods using microorganisms, methods based on microorganisms, etc., can solve the problems of weakening the virulence of streptococcus agalactiae derived from tilapia, and it is not easy to achieve Effects of returning poison and weakening poison

Active Publication Date: 2016-02-03
GUANGXI ACADEMY OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] Aiming at the difficulties in the development and application of tilapia streptococcus agalactiae disease vaccine, the main problem to be solved by the present invention is to provide a method for weakening the virulence of tilapia-derived streptococcus agalactiae, and to obtain attenuated streptococcus agalactiae from tilapia. Strain for the development of attenuated vaccine against tilapia streptococcal disease

Method used

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  • A method for weakening the virulence of streptococcus agalactiae derived from tilapia
  • A method for weakening the virulence of streptococcus agalactiae derived from tilapia
  • A method for weakening the virulence of streptococcus agalactiae derived from tilapia

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Embodiment 1

[0024] Embodiment 1 The culture medium that the present invention uses

[0025] Tryptone Soy Broth (TSB): tryptone 17.0g / L, soybean peptone 5.0g / L, NaCl 5.0g / L, glucose 2.5g / L, distilled water 1000mL, adjust pH to 7.2-7.4, 121℃ under high pressure Sterilize for 20 minutes.

[0026] Low nutrient tryptone soybean broth (LTSB): tryptone 9.0g / L, soybean peptone 2.5g / L, NaCl 5.0g / L, glucose 2.5g / L, distilled water 1000mL, adjust pH 7.2-7.4, 121 ℃ autoclave for 20min.

Embodiment 2

[0027] Example 2 Rapid culture of tilapia source Streptococcus agalactiae wild virulent strain HN016 by temperature difference passage

[0028] Isolation of wild strains of Streptococcus agalactiae from tilapia: Use a bacterial inoculation loop to inoculate and isolate the brain, liver, kidney, spleen or fins of tilapia with large-scale disease-affected farms, and culture at 28°C for 24 hours. Pick a single colony and expand it in TSB liquid medium for 24 hours. After microscopic examination without pollution, carry out virulence determination and PCR diagnosis. After confirming that it was Streptococcus agalactiae, the bacteria solution and glycerin were mixed thoroughly at a ratio of 4:1 (V / V), and stored at -80°C.

[0029] The wild virulent strain HN016 (LD) stored at -80°C 50 =7.08×10 3 CFU / tail) was streaked on a blood plate, and a single colony was picked and cultured in a 50mL culture tube containing 20mL TSB medium at 25°C with shaking. Step 1: After culturing for 6...

Embodiment 3

[0033] Example 3 Virulence Comparison of Tilapia Source Streptococcus agalactiae Wild Strain HN016 Before and After Attenuation of Virulence

[0034] After the original and weakened strains of the strong wild strain HN016 were cultured with TSB for 24 hours, the cultured bacteria were used to infect tilapia by intraperitoneal injection and oral infection, respectively. Inject infection by stock solution (1×10 9 CFU / mL), 1:2, 1:10, 1:100, 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 For dilution challenge, each fish was injected with 0.2mL culture solution, 20 fish / group, the water temperature was 30-31.5°C, the size of the test fish was 25g / tail, and observation and records were made for 30 days. The results of injection infection showed that the wild virulent strain culture liquid stock solution, 1:2, 1:10, 1:100, 1:10 3 , 1:10 4 The mortality rate of the diluted virus was more than 50%. 24 hours after the challenge, the typical symptoms of streptococcal disease such as cloudy and...

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Abstract

The invention discloses a method for weakening the toxicity of tilapia sourced streptococcus agalactiae. The method disclosed by the invention comprises the following steps of carrying out streak culture of a separately stored wild strain on a blood plate, and picking single colonies to a TSB culture medium to carry out shake culture at 25 DEG C; step 1, after culturing for 6 h, inoculating single colonies on a novel TSB culture medium to carry out shake culture at 25 DEG C, and subcultring every 6 h till 20 generations; step 2, inoculating the 20 generation of culture on a novel LTSB culture medium to carry out shake culture at 35 DEG C, and subcultring every 6 h till 40 generations; processing the 41 generation of culture by repeating the step 1 till processing the 60 generation of culture, and processing the 61 generation of culture by repeating the step 2 till processing the 80 generation of culture, wherein subcultring is continuously carried out according to the method; the toxicity of the strain is tested in the subcultring process, and subcultring is carried out till the toxicity of the strain is weakened. The method for weakening the toxicity of tilapia sourced streptococcus agalactiae is simple and rapid; the method for weakening the toxicity of a tilapia sourced streptococcus agalactiae wild strain is weak in toxicity and difficultly becomes toxic; the method has important significance and application value for developing attenuated vaccines of tilapia streptococcicosis.

Description

technical field [0001] The invention relates to a method for weakening the toxicity of streptococcus, in particular to a method for weakening the toxicity of streptococcus agalactiae derived from tilapia. Background technique [0002] Streptococcus widely exists in nature, humans and animals, causing various suppurative inflammations, scarlet fever, erysipelas, neonatal sepsis, meningitis, puerperal fever and streptococcal allergic diseases. Aquaculture animals can also be infected with Streptococcus and cause streptococcal disease, causing mass mortality and causing huge economic losses to the world's aquaculture industry every year. my country is a big aquaculture country, and the direct economic loss caused by streptococcal disease to aquaculture is about 1 billion US dollars every year. Tilapia is an important tropical and subtropical aquaculture animal and one of the main targets of streptococcal infection. my country is the largest country in tilapia farming, and its...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/36C12R1/46
Inventor 李莉萍王瑞陈明甘西梁万文黄婷朱佳杰雷爱莹李健陈福艳
Owner GUANGXI ACADEMY OF FISHERY SCI
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