A Study On Screening And Practicality Of Bacteriophage Of Aeromonas Veronii From Pelteobagrus Fulvidraco

As a well-known and excellent breed,Pelteobagrus fulvidraco is favored by breeding producers because of its easy feeding,high and stable breeding profits,and consumers’love.In recent years,under the conditions of intensive and high-density aquaculture,the outbreak of Aeromonas veronii disease has caused huge economic losses to the Pelteobagrus fulvidraco industry and become one of the bottlenecks restricting the development of the Pelteobagrus fulvidraco industry.The over-dependence and abuse of antibiotics by farmers has led to an increase in the resistance of pathogenic bacteria,resulting in a greatly reduced effect of antibiotic use,and it is urgent to find a new type of substitute agent.In this study,a strain of pathogenic Aeromonas veronii was isolated from the diseased Pelteobagrus fulvidraco,and the phages were screened as the host bacteria,and the phages with good bacteriostatic effect and able to protect the Pelteobagrus fulvidraco against Aeromonas veronii infection were further selected from the phages isolated and purified,and finally the biological characteristics of the above phages were studied as follows:1.Isolation and identification of Aeromonas veronii and isolation of bacteriophages in Pelteobagrus fulvidracoA dominant strain was obtained from the muscle ulceration,liver,kidney and spleen tissues of a Pelteobagrus fulvidraco with muscle ulcer disease in Shashi District,Jingzhou City,Hubei Province,named ZYAH128,and confirmed that ZYAH128 was confirmed as Aeromonas veronii by morphological observation,physiological biochemical identification,and phylogenetic tree construction.Regression infection experiments were subsequently conducted to confirm its pathogenicity,and the LD50of Aeromonas veronii ZYAH128 was 6.31×10~5 CFU/m L under the Pelteobagrus fulvidraco scratch soaking infection model.Drug susceptibility tests found that Aeromonas veronii ZYAH128 was 100%resistant to sulfonamides and 100%sensitive to quinols and amide alcohols.Finally,ZYAH128 was used as the host bacteria to screen for Aeromonas veronii bacteriophages,and a total of 33 strains of bacteriophages were screened.Further,33 bacteriophages were lysed,and 11 strains of Bacteriostematoca microbacterium ZPAV-1,ZPAV-16,ZPAV-18,ZPAV-20,ZPAV-21,ZPAV-22,ZPAV-23,ZPAV-24,ZPAV-25,ZPAV-26 and ZPAV-27 were screened,and the above phages were able to inhibit the growth of ZYAH-128.2.Preliminary application and biological characteristics of Aeromonas veronii phageThe 11 phages obtained by screening were subjected to the prevention and treatment experiment of Aeromonas veronii infection,10 LD50 was selected as the experimental attack concentration,and the phages were treated with pre-infection prevention and post-infection treatment according to MOI=1,respectively,and finally two strains of phages with excellent protective effect were obtained,ZPAV-18 and ZPAV-25,ZPAV-18 prevention protection rate was 85.7%,treatment protection rate was71.4%,ZPAV-25 prevention protection rate was 85.7%,The therapeutic protection rate was 85.7%.Biological characteristics studies have shown that ZPAV-18 and ZPAV-25phages are lytic bacteriophages,and transmission electron microscopy has found that the two phages have tailed phages with regular polyhedron on their heads.ZPAV-18 has an optimal infection complex of 0.01,which can maintain high activity in the range of20°C to 60°C,and can still maintain high activity under 30 min treatment at 70°C and80°C,and can tolerate p H 3-12;99.7%of the phages within 15 min are adsorbed on the host bacteria;the incubation period is 15 min,the growth phase is 35 min,and the platform phase titer reaches 108 PFU/m L.ZPAV-25 has an optimal infection complex of 0.001,maintains high activity in the range of 20°C-80°C,and can tolerate p H 3-12;98.3%of bacteriophages within 15 min are adsorbed on the host bacteria;the incubation period is 20 min,the growth period is 40 min,and the platform phase titer reaches 10~7PFU/m L.In summary,the above-mentioned phages show good temperature and p H tolerance,fast adsorption rate,high lysis efficiency,and have the potential to develop into biological control preparations for Aeromonas veronii in Pelteobagrus fulvidraco,and this study also provides a reference for the development of prevention and control strategies for other fish bacterial diseases.