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Attenuated Strain of Streptococcus agalactiae from Tilapia and Its Application

A technology of streptococcus lactis and tilapia, applied in the direction of bacteria, antibacterial drugs, bacterial antigen components, etc., can solve the problems of high cost of immune prevention and control, short immune protection period, inconvenient immune route, etc., and achieve good immune protection effect , protection against infection, and highly effective immune protection

Active Publication Date: 2016-09-28
GUANGXI ACADEMY OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problems of high cost of immunization prevention and control of tilapia streptococcosis inactivated vaccine, short immunization protection period, inconvenient immunization route, etc., to provide attenuated strains of Streptococcus agalactiae derived from tilapia and its application, and to develop tilapia Streptococcus disease attenuated live vaccine

Method used

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  • Attenuated Strain of Streptococcus agalactiae from Tilapia and Its Application
  • Attenuated Strain of Streptococcus agalactiae from Tilapia and Its Application
  • Attenuated Strain of Streptococcus agalactiae from Tilapia and Its Application

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Embodiment 1

[0013] Embodiment 1 Bacterial strain and medium used in the present invention

[0014] The wild type strain HN016 of Streptococcus agalactiae derived from tilapia was isolated from the brain of a tilapia farm where streptococcal disease broke out in southern my country. ).

[0015] The tilapia-derived Streptococcus agalactiae attenuated strain YM001 was obtained by attenuating the wild-type strain HN016 by using the rapid culture and attenuating method of temperature difference subculture. It is preserved in the China Center for Type Culture Collection CCTCC, and the preservation number is: CCTCCM 2014045.

[0016] 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, autoclave at 121 °C for 20 min, cooled at room temperature for later use.

Embodiment 2

[0017] Example 2 PCR and biochemical identification of tilapia-derived Streptococcus agalactiae attenuated strain YM001

[0018] The attenuated strain YM001 was identified by PCR identification method of tilapia-derived Streptococcus (Streptococcus iniae and Streptococcus agalactiae), API 20 Strep biochemical strip and VITEK 2 automatic microbial analyzer.

[0019] Double PCR rapid detection of Streptococcus iniae and Streptococcus agalactiae: PCR primers refer to published papers by Li Jiong et al. (2010) and Zlotkin et al. (1998). The size of the fragment amplified by cocci-specific primers was 296 bp. The PCR reaction reagent is a PCR kit from TaKaRa Company in Dalian, China, and the PCR system is as follows: 10× Ex Taq Buffer (Mg 2+ ) 5.0 µL, dNTP 2.0 µL, H1, H2, P1 and P2 primers 2.0 µL each, 2.0 µL DNA template (approximately 50 ng), 1.0 µL Takara Ex Taq (5 U / µL), 32.0 µL ddH 2 O, total volume 50 µL; PCR reaction was carried out on GeneAmp PCR system 9700 from ABI...

Embodiment 3

[0022] Example 3: The median lethal dose LD of tilapia-derived Streptococcus agalactiae wild-type strain HN016 and attenuated strain YM001 to tilapia 50 determination

[0023] Use TSB to culture tilapia-derived wild-type strain HN016 and attenuated strain YM001, respectively, at 25-30 °C, shake the shaker for 18-24 hours, and use the plate colony counting method to count the bacteria in the liquid. Eight serial dilutions of the culture solution were performed with a 10-fold gradient, and 20 tilapias were infected by intraperitoneal injection of each concentration of the bacterial solution, 0.1 ml / tail; the same volume of TSB medium was injected intraperitoneally into tilapias in the control group, 0.1 ml / tail . Both the infected group and the control group were kept in an inflatable aquarium with 40 L of water, 2 / 3 of the water was changed every 2 days, the water temperature was controlled at 30-32 ℃, and the fish were fed twice a day in the morning and afternoon based on 3%...

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Abstract

The invention relates to the disease prevention and control technology of aquaculture animals, in particular to the attenuated strain of Streptococcus agalactiae derived from tilapia and its application. The attenuated strain of Streptococcus agalactiae derived from tilapia is YM001, which is preserved in CCTCC, and the preservation number is: CCTCC M 2014045. After the attenuated strain was activated and fermented, the 10 6 -10 9 Bacterial cells of the attenuated strain YM001 of CFU / mL are mixed with sterile phosphate buffer solution as an attenuated live vaccine for the prevention and treatment of tilapia streptococcosis, and the injection concentration is 10 5 -10 8 CFU / tail, oral administration concentration is 10 8 -10 9 CFU / tail, soaking administration concentration is 10 7 -10 8 CFU / mL. The invention relates to strain identification, virulence measurement, safety and stability of reversion, immunogenicity and immune protection effect of the attenuated strain. The tilapia streptococcal disease vaccine made with the strain has good immune protection effect and safety and strong stability.

Description

technical field [0001] The present invention relates to aquaculture animal disease control technology, specifically relates to Streptococcus agalactiae ( Streptococcus agalactiae ) infected tilapia streptococcosis immune control attenuated strain and its application. Background technique [0002] my country is the largest country in tilapia farming, and its annual output accounts for more than 50% of the world's total output. From 2001 to 2008, streptococcal infection occurred successively in tilapia farming in China, and caused a high mortality rate of 30% to 50% in individual farms. The main epidemic strain was Streptococcus iniae; 2009 to 2013 for 5 consecutive years , streptococcal disease has broken out in a large area of ​​tilapia aquaculture in China, and the incidence area is expanding year by year, and the morbidity and mortality are increasing year by year. et al., 2011; Maixin Lu et al., 2010; Zhu Jinglin et al., 2010; Zhou Suming, 2008). Calculated according...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A61K39/09A61P31/04C12R1/46
Inventor 陈明李莉萍王瑞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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