Edwardsiella tarda natural low virulent strain and application thereof
A weak strain and blunt technology, applied in the direction of bacteria, antibacterial drugs, bacterial antigen components, etc., to achieve the effect of eliminating possibility, improving environmental safety and controllability, and good control effect
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Embodiment 1
[0025] The preparation of embodiment 1 attenuated live vaccine
[0026] (1) Isolation and identification of natural attenuated strains of Edwardsiella tarda
[0027] In 2008, a series of Edwardsiella tarda were isolated from the liver, spleen and kidney of turbot suffering from Edwardsiella in Yantai, Shandong Province. The spleen, liver and ascites of 5 diseased turbot were mixed and homogenized respectively, and after gradient dilution, they were spread on gallic acid milk agar (DHL), cultured overnight at 30°C, and the system identification was carried out after a single colony grew out.
[0028] Most of them grew black-core colonies unique to Edwardsiella tarda, and 30 characteristic single clones were picked and named EIB101-EIB310 respectively, and the species were preserved for subsequent systematic identification. Two single clones without black hearts were found on the spleen sample coating plate, and the single clones were picked and re-stretched separately, and it ...
Embodiment 2
[0040] Embodiment 2: Detection of pathogenicity of Edwardsiella tarda natural attenuated strain EIBAV1
[0041] Pathogenicity assays were performed on different target animals.
[0042] (1) Detection of pathogenicity of attenuated EIBAV1 strain on zebrafish
[0043] Under laboratory conditions, zebrafish was used as a model animal to detect the virulence of the isolated natural attenuated strain EIBAV1 of Edwardsiella tarda. The fish used in the test were first placed in a clean tank to adapt to breeding for 1 week, so as to remove abnormal individuals. Before the infection test, the fish for the health test were cultured in a 0.5L infection test tank, and fed for 1 week, and 10 fish were cultured in each tank (average body length 2.5-3cm, body weight 0.2g). The test tank replaced 1 / 2 volume of culture water with sterile old water every day, and the water temperature was 28°C, with a fluctuation of 2°C.
[0044] The fish used in the test were randomly divided into groups, a...
Embodiment 3
[0062] Embodiment 3: Evaluation of immune protection rate of Edwardsiella tarda natural attenuated strain EIBAV1 in different animal models
[0063] (1) Immune protection test using zebrafish as experimental animal
[0064] The experimental zebrafish were randomly divided into 3 groups, each with 3 parallel tanks, 20 fish per tank. The attenuated strain EIBAV1 was used as a live vaccine to immunize zebrafish by intramuscular injection, and the injection dose was 10 4 CFU / g. The control group was injected with sterile normal saline. One month after the immunization, the zebrafish in each group were artificially infected with the wild strain EIB202 of Edwardsiella tarda. Observe and count the death numbers of the control group and the immune group within 15 days, and calculate the immune protection rate of each group (see Table 4).
[0065] Among them, the immune protection rate was calculated according to the following formula: relative immune protection rate (RPS)%=(mortal...
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