Screening Of Aeromonas Salmonicida Strain And Identificaiton Of Its Cytotoxin

Aeromonas salmonicida is one of the main pathogens both in freshwater and marine fishes.Not only had it caused huge economic losses to the United States and European countries,but also this bacterial pathogen had affected aquaculture industry in many Asian countries including China.Screening of Aeromonas salmonicida strains,identifying its toxins,and studying the pathogenic mechanism play an important role in the prevention and treatment of Aeromonas salmonicida.In this study,a gram-negative bacteria was isolated and purified from the intestine of grass carp by the means of streak plate.Based on the morphological,physiological and biochemical characteristics of the strain and 16 S r DNA sequence analysis,it was identified as Aeromonas salmonicida and named DBFF01.The morphology,growth characteristics and antibiotic sensitivity of DBFF01 strain were analyzed in this research.Through the whole genome sequencing,the samples were analyzed for data statistics and quality control,including the plasmids sequences analysis,base composition and Coverage statistics and repeat sequence analysis and so on.Then,the gene structure and function annotation had been done according to the sequencing results,including the prediction of t RNA and r RNA,protein length,COG,KEGG databases and so on.The results showed that the total length of the genome was 794,967,393 bpand the number of reads was 59,244.It was found that the average length of reads was 2,950 bp,and the GC% was 58.33%.What's more,there were 10 ribosomal r RNA operons and 129 t RNAs were identified.Most of the genes were related to carbohydrate metabolism,amino acid metabolism and substance transport according to the COG and KEGG databases.There was a gene homoeologous with the B.thuringiensis subsp.israelensis Cyt1 Aa toxin compared to the SWiss Prot database.This gene which was cloned was induced to express the protein and was purified before it was incubated to the midgut cell lines CF203/2.5 from Choristoneura fumiferana.It was found that the cells were aggregated and ruptured over time.In addition,the number of cells decreased continuously compared with the control group.The cytotoxicity was increased gradually with the time and concentration increased.The results showed that the protein was toxic to CF203/2.5 cells,the reasons why it was toxic to insect cells need to be study in the future.In this research,the screening,identification and sequencing of A.salmonicida DBFF01 strain were carried out and the potential toxin proteins were cloned and expressed and used in cytotoxicity test.All of this work is beneficial to elucidate the pathogenic mechanism of A.salmonicida.It provides a theoretical basis for the prevention and treatment of furunculosis.