| As the culture density of Litopenaeus vannamei increasing and abuse of theantibiotics, the traditional treatments of diseases caused by Vibrio alginolyticusbecome more and more difficult. It need a new method of treatment for diseases. Thecurrent study included separation of Vibrio alginolyticus and its bacteriophage;preparing vaccines of bacterial ghost from its bacteriophage; production of Vibrioalginolyticus inactivated vaccine by formalin; extraction of lipopolysaccharide byphenolic water method and the effects of disease resistance of the Litopenaeusvannamei which were immune with these three preparations by injection or feedingwere observed. The main contents and conclusions are as follows:1. A stain of bacteria which isolated from drainage of the Aquatic Products WholesaleMarket (APWM) in Zhanjiang city was confirmed as Vibrio alginolyticus usingbiochemical identification and examination of the hypervariable region of ToxR geneby PCR.2. A strain of bacteriophage carried by the Vibrio alginolyticus was isolated fromAPWM. The diameter of the bacteriophage plaque was1-1.5mm (12h). Thebacteriophage was UV-sensitive. The lytic activity of the bacteriophage lost within30min as it was exposed to UV light (20W,30cm). It had stronger lytic activity at pH8-11, and the optimum pH was11. The lytic cycle was65min. It was sensitive totemperature above65℃. It was tolerated to ether and chloroform. It was sensitive toantiviral drugs acyclovir. The maxima routine test dilution (RTD) was106PFU/mL.The optimal multiplicity of infection was10.3. Adding Formalin to the culturing liquid of Vibrio alginolyticus after12h to finalconcentrations of0.1%,0.2%,0.4%,0.6%and0.8%, the mixtures were kept at4℃,25℃and35℃for inactivation, and placed in a cyclotron oscillator at25℃forinactivation. Then the efficiency of the inactivation was detected by spread plate method. Screened the ideal inactivating condition was adding0.1%formalin andshaking at25℃for12h. The production rate of LPS extracted fromVibrioalginolyticus with phenolic water was0.7529%. Its protein and polysaccharidecontents were22.1μg/mg and190.7μg/mg separately. The Litopenaeus vannamei (7-9g) injected5mg/mL,4mg/mL,3mg/mL,2mg/mL,1mg/mL,0.5mg/mL LPS separately,and the injection volume was0.1mL. The results showed3mg/mL LPS was lethal toLitopenaeus vannamei.4. The methods of preparation of bacterial ghost of Vibrio alginolyticus frombacteriophage: Vibrio alginolyticus was washed by sterile seawater, put it togetherwith concentration bacteriophage liquid by proportion of MOI=10, then adding sterileseawater and response for6h. According to the number of bacteria number and itsnucleic acid concentration in supernatant by centrifugation had linear relationship,we got the linear equation y=8E+06x-4E+06, within2.5-19.7μg/mL nucleic acidconcentration had a good linear relationship. This could be used to calculate thevaccines concentration of bacterial ghost. The bacterial ghost of Vibrio alginolyticusadded garlic liquid by1:1and reacted for3.5h to remove residual bacterium. At lastthe bacterial ghost was washed repeatedly with PBS.5. The Litopenaeus vannamei was immune with inactivation bacterial ghost, vaccineand LPS by injection; and was immune by feeding the bacterial ghost directly. Thedifference between the three method was not obvious (P<0.05). The Litopenaeusvannamei injected with1×107cell/mL bacterial ghost, Relative Percent of Survival(RPS) was higher within24h, and the RPS got it maxima at53.56%after12h. TheLitopenaeus vannamei injected1×107cell/mL Vibrio Alginolyticus intramuscularly, themaxima RPS was57.14%after immunization48h. The RPS at a concentration of0.5mg/mL LPS was57.14%after immunization for24h.The Litopenaeus vannamei was immune with bacterial ghost, the RPS could beincreased by elongation of time. The Litopenaeus vannamei was feeded with1×109cell of bacterial ghost per gram of the dietary, the maxima RPS was28.57%after immunization48h.6. By observation of the proportion of blood lymphocytes, the activity of PO in serumand the phagocytic of blood cells,the bacterial ghost and vaccine increased the POactivity by promoting of the proliferation of Granular cells. LPS increased the POactivity by promoting the proliferation of Small granular cells, but it would also havea strong toxic effect at higher concentration. 7. The enhancement effect of vaccines and bacterial ghost on the SOD activity isbetter than LPS.8.Litopenaeus vannamei non-specific immunity would be enhanced by bacterial ghost... |
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