Method for weakening toxicity of tilapia sourced streptococcus agalactiae

A technology for Streptococcus lactis and tilapia, applied in biochemical equipment and methods, methods using microorganisms, and methods based on microorganisms, etc. The effect of returning poison and weak poison

Active Publication Date: 2014-06-25
GUANGXI ACADEMY OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 weakenin

Method used

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  • Method for weakening toxicity of tilapia sourced streptococcus agalactiae
  • Method for weakening toxicity of tilapia sourced streptococcus agalactiae
  • Method for weakening toxicity of tilapia sourced streptococcus agalactiae

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1 The medium used in the present invention

[0025] Tryptone Broth (TSB): tryptone 17.0 g / L, soybean peptone 5.0 g / L, NaCl 5.0 g / L, glucose 2.5 g / L, distilled water 1000 mL, adjust pH to 7.2-7.4, autoclaved at 121 ℃ Bacteria for 20 min.

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

Embodiment 2

[0027] Example 2 Rapid culture of tilapia-derived 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 . A single colony was picked and cultured in TSB liquid medium for 24 hours. After microscopic examination without contamination, the virulence test and PCR diagnosis were carried out. After it was confirmed as Streptococcus agalactiae, the bacteria solution and glycerin were mixed thoroughly at a ratio of 4:1 (V / V), and stored in a freezer at -80°C.

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

Embodiment 3

[0033] Example 3 Comparison of virulence before and after weakening virulence of wild strain HN016 of tilapia-derived Streptococcus agalactiae

[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 0.2 mL culture solution, 20 fish / group, the water temperature was 30-31.5 ℃, the size of the test fish was 25 g / 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 streptococcus disease such as clo...

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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 present invention involves a weakening method of poisoning force for a splin bacteria, which specifically involves the weakening method of poisoning power for the lactococcus without milk. Background technique [0002] Chain bacteria exist widely in nature, human and animals, causing various purulent inflammation, scarlet fever, van poison, neonatal sepsis, meningitis, puerperic fever, and chain -perverted response diseases.Aquaculture animals can also infect link bacteria, causing large -scale deaths, causing huge economic losses to the world's aquaculture industry each year.my country is a major aquaculture country. It is about $ 1 billion in direct economic losses caused by split bacteria to aquaculture every year.Luo Feiyu is an important tropical and subtropical aquaculture animals, and is also one of the main objects of Chainbococcal infection.my country is the largest country of Luo Fei Fish breeding, with an annual output accounting for more tha...

Claims

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Application Information

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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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