| Vibrio vulnificus is a mesophilic and halophilic marine pathogen with a wide distribution,mostly in coastal areas,and can be found in seawater,seabed sediments and marine organisms.Vibrio vulnificus can not only make a variety of economic animals in aquaculture sick,such as tilapia,golden pomfret,eel,sturgeon,grouper,grass carp and white shrimp,etc.,but also threaten human life and health through consumption and wound infection,which has a very negative impact on the global aquaculture industry.At present,the vaccine of Vibrio vulnificus is still in the laboratory research and development stage.The breeding industry in my country is mainly based on antibiotics.However,the abuse of antibiotics will lead to the problem of drug resistance of pathogenic bacteria and drug residues.It is urgent to find a green and safe antibiotic.alternatives.Immunoglobulin of yolk(Ig Y)is an immunoglobulin mainly present in the serum of birds,amphibians and reptiles.Ig Y has the characteristics of high yield,low cost,stable physical and chemical properties,etc.,and has been widely used in the prevention and treatment of human,animal husbandry,and aquaculture diseases.In this study,inactivated Vibrio vulnificus was used as an antigen to immunize laying hens,and anti-V.vulnificus Ig Y were prepared.as follows:(1)Ig Y extraction and purification and titer detection.The collected egg yolk solution of Wumen was separated and purified by water dilution-freezing-thaw-ammonium sulfate secondary salting-out-dialysis-ultrafiltration concentration method,and its titer was as high as 1:51200 as determined by ELISA method.(2)Stability analysis of egg yolk antibody.The stability of anti-V.vulnificus egg yolk antibody was measured by indirect ELISA,and the relative activity could be maintained under the conditions of below 65°C,p H=2～8,and pepsin digestion(p H=2～8),proving that anti-trauma Vibrio yolk antibody has the characteristics of high temperature resistance,acid and alkali resistance,and pepsin digestion resistance.(3)Direct effect on Vibrio vulnificus.Through bacterial agglutination experiments,it was proved that the anti-V.vulnificus Ig Y can specifically recognize and act on Vibrio vulnificus.The effect of specific yolk antibody on the biofilm formation ability of Vibrio vulnificus was detected by crystal violet staining quantitative method.The results showed that the specific yolk antibody at a concentration higher than 5 mg/m L could prevent the adhesion of Vibrio vulnificus and affect the biofilm formation.(4)The preventive and protective effect on zebrafish.The survival rate of the experimental group injected with anti-V.vulnificus Ig Y was 90%,which was significantly higher than that of the challenge experimental group(40%)(p<0.05),and the relative protection rate was 83.3%.The relative protection rates of the 10‰,5‰,and 1‰ yolk antibody feed groups were 71.43%,28.57%,and 14.29%,respectively.The survival rate(80%)of the 10‰ yolk antibody feed group was significantly higher than that of the common feed group.(30%)(p<0.05).There was no significant difference in the survival rate of the experimental group soaked with 10‰ and 1‰ egg yolk antibody compared with the challenge control group(p>0.05).(5)Effects on the intestinal flora of zebrafish.Beta diversity analysis between samples was used to analyze the similarity of species diversity between different samples.The species diversity of blank control group,Ig Y injection group,and Ig Y prevention group was far from the model group.On the species abundance accumulation map at the genus level,the proportion of Vibrio in the Ig Y prevention group was 7.24%,which was much lower than the proportion in the model group,the proportion in the model group was 31.76%,the blank control group and Ig Y The proportion of Vibrio genus in the injection group was 0.07% and 0.04%,and the content was extremely low,which indicated that the anti-V.vulnificus Ig Y could effectively inhibit the growth and reproduction of Vibrio vulnificus in the zebrafish intestine.In conclusion,the yolk antibody prepared by using Vibrio vulnificus as an antigen in this study has a good preventive and protective effect on infected zebrafish,and provides a certain scientific basis for the in-depth study of the bacteriostatic mechanism of Ig Y and the disease prevention and control of Vibrio vulnificus. |
