Supervirulent df-1 cell-adapted strain of chicken infectious bursal disease and its application
A technology of bursal disease and chicken infectivity, applied in the field of animal virology, can solve problems such as increased diagnosis and treatment, poor sanitation, chaotic feeding management, etc., and achieve good immunogenicity, stable efficacy, and high safety Effect
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Embodiment 1
[0015] Example 1 Isolation and Identification of Infectious Bursal Virus CK / SD / LY / 2014 Strain
[0016] In August 2014, a suspected IBD epidemic occurred in a chicken farm in Linyi, Shandong. The bursa and spleen tissues of sick chickens with typical symptoms and pathological changes were aseptically collected, chopped and ground, and weighed according to the weight ratio of 1:3. Add sterilized PBS, centrifuge at 4°C and 6000r / min for 10min, centrifuge at 4°C and 8000r / min for 10min, transfer the supernatant to another sterile 1.5ml centrifuge tube, add double antibody (final concentration of penicillin 2000IU / ml, streptomycin 2mg / ml), act at 37°C for 1 hour, inoculate 5 10-day-old SPF chicken embryos through the allantoic membrane, 0.2mL / piece. Discard the chicken embryos that died within 24 hours, observe once every 6 hours thereafter, collect the dead chicken embryos, take out all the allantoic membranes and embryo bodies of the chicken embryos at 144 hours, mash the tissue...
Embodiment 2
[0019] Example 2 Determination of biological characteristics of infectious bursal virus CK / SD / LY / 2014 strain
[0020] Chicken embryo inoculation test:
[0021] Inoculate 10 9-10-day-old SPF chicken embryos with 0.2 mL of the above E1 virus supernatant by chorioallantoic membrane (CAM) inoculation method, and set the same dose of sterile PBS as the negative control group. Place the eggs in a 37°C incubator to continue incubating. Check the eggs every 12 hours, discard the dead embryos within 24 hours, record the death and pathological changes of the chicken embryos, and collect all surviving chicken embryos and observe the pathological changes after 144 hours of inoculation observation. The chicken embryos died within 48-72 hours after inoculation. The allantoic membrane of the dead embryos was thickened, and the abdomen was swollen. The autopsy showed that the liver of the chicken embryos had yellow degeneration, accompanied by mottled necrosis, which was consistent with the l...
Embodiment 3
[0035] Example 3 Screening and identification of cell-adapted strains
[0036] The E1 generation CK / SD / LY / 2014 virus liquid was blindly transferred to the E5 generation with chicken embryos. DF-1 cells were divided into 3×10 5 The dose per well was spread on a 6-well plate, and the E5 generation virus solution was diluted 10 times in DMEM to 10 -4 ~10 -8 . Rinse the DF-1 cells grown to a monolayer twice with serum-free DMEM, then inoculate the diluted virus solution on the DF-1 cells, inoculate 3 wells for each dilution, 0.3mL / well, absorb at 37°C After 1.5h, discard the remaining virus liquid. After that, add the first layer of nutrient agar about 3 mm thick, and place in CO 2 Incubator, 37°C, 5% CO 2 After upside-down culture for 48 hours under the conditions (when the cells become lesions), add a second layer of nutrient agar containing 0.01% neutral red, 37 ° C, 5% CO 2 Cultured upside down in the dark. After the plaques were formed, suck them out with a dropper, p...
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