High pathogenicity Aeromonas hydrophila and application
An Aeromonas hydrophila, highly pathogenic technology, used in bacteria, antibacterial drugs, veterinary vaccines, etc., to reduce economic losses, enhance immunity, and prevent bacterial sepsis
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Embodiment 1
[0031] Acquisition of a highly pathogenic Aeromonas hydrophila:
[0032] (1) Screening of highly pathogenic Aeromonas hydrophila with multiple virulence factors
[0033] A. Dissect the diseased or dying fish in an ultra-clean bench, streak from the diseased part onto TSB solid medium, culture at 28°C for 18h-24h, pick a single colony on the plate and transfer it to fresh medium for pure culture ; Typical colonies on TSB solid medium are round, light yellow, translucent, smooth, moist, and neatly bordered colonies.
[0034] B. Get the single colony of pure culture on the above-mentioned plate, carry out biochemical identification (see Table 1) with the API bacteria identification reagent strip of France Mérieux Company, and carry out Gram staining and observe bacterial colony morphology.
[0035] Table 1: Partial physiological and biochemical identification results of isolates
[0036]
[0037] C. The 16SrDNA of the candidate strain was amplified and sequenced, and the hom...
Embodiment 2
[0054] Recovery and growth curve of Aeromonas hydrophila ZYAH72:
[0055] Cultivation of Aeromonas hydrophila ZYAH72: Take out the strain stored in glycerin from the -80 degree refrigerator, streak it on the TSB solid medium, cultivate it in a 28°C incubator for about 24 hours, and then pick a single colony on the plate Streak culture on fresh TSB solid medium, after subculture and recovery twice, pick a single colony from TSB solid medium and inoculate it in TSB liquid medium, then culture at 28°C, 180r / min shaker, every 1 Hourly measure its OD 600nm Absorbance value, the same operation is applied to the pathogenic standard strain, the results show that both ZYAH72 and the pathogenic standard strain enter the logarithmic phase at about 2h, enter the plateau phase at 4h-5h, and contain about 10 8 ~10 9 CFU / mL, the absorbance value of ZYAH72 will be higher than that of the pathogenic standard strain after entering the stationary phase, indicating that the growth rate of the i...
Embodiment 3
[0057] Preparation of Aeromonas hydrophila ZYAH72 inactivated vaccine:
[0058] Selection of optimal formaldehyde concentration:
[0059] Add formaldehyde solutions with a final concentration of 0.1%-0.8% (volume fraction) to the cultured bacteria solution to inactivate the highly pathogenic Aeromonas hydrophila, and continue culturing in a shaker at 28°C for 12 hours , different inactivated vaccines were coated on TSB solid medium, and the results showed that complete inactivated vaccines could be prepared when the formaldehyde concentration was 0.3%-0.8%. %, the antigen loss of the inactivated vaccine is the smallest, and the inactivated vaccine prepared with this formaldehyde concentration is used as the immune vaccine.
[0060] Preparation of inactivated vaccine:
[0061] The isolated highly pathogenic Aeromonas hydrophila ZYAH72 was rejuvenated for 2 generations on the Columbia blood plate, and a single colony on the blood plate was picked and inoculated in TSB medium, ...
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