Tissue Distribution And Adhesion Function Of Apostichopus Japonicus Infected By Vibrio Splendidus

Vibrio splendidus is the pathogen of many kinds of marine animals,which can cause many diseases of fish,shellfish and so on.V.splendidus is also the main pathogen of Apostichopus japonicus,which causes the skin ulcer syndrome.Understanding the dynamic distribution of pathogenic bacteria in the host is an important part of the pathogenic mechanism of pathogenic bacteria,which provides a basis for the study of the pathogenesis and pathological mechanism of diseases,and is of great significance for the diagnosis,prevention and control of diseases.Based on the tissue distribution and adhesion process of A.japonicus infected by V.splendidus,the following research results have been achieved in this project:（1）In this study,we found the dynamic distribution of V.splendidus in the body of A.japonicus through the V.splendidus tagging method and infection experiment.In the early infection stage of 6 h,more V.splendidus was detected in the body wall and tentacle tissues.With the infection time to 96 h,the content of V.splendidus in all tissues of A.japonicus increased first and then decreased.The highest detection amount in the respiratory tree and intestinal tissues can respectively reach 6.8×10⁶CFU/g and 1.1×10⁷CFU/g.The highest detection amount in coelomic fluid reached 2.6×10¹⁰CFU/m L.According to the results,it was inferred that the respiratory tree,intestinal tissue and the coelomic fluid are the main adhesive sites of V.splendidus.In the experiment of V.splendidus infecting the coelomocytes of A.japonicus,we found that V.splendidus exists as an extracellular bacteria,and proved that its adhesion to the surface of coelomocytes requires the participation of the surface recognition receptorβ-integrin of A.japonicus but it rarely internalized into coelomocytes.（2）NCBI Genome sequencing showed that there are two homologous Dihydrolipoamide dehydrogenase（DLD）genes in V.splendidus.In this experiment,we cloned the DLD1 and DLD2of V.splendidus,and obtained the active DLD1 and dld2 proteins through prokaryotic expression The enzyme activity of DLD1 and DLD2 was determined.DLD1 and DLD2 both belong to NADH oxidase family.The antibodies against DLD1 and DLD2 were prepared.It was confirmed by Whole cell ELISA that DLD1 and DLD2 both located on the surface of V.splendidus.Study on the adhesion function of DLD1 and DLD2:Through the antibody blocking experiment,it was found that the adhesion of V.splendidus to the climbing plate,the coelomocytes and the individuals of A.japonicus was significantly reduced.By using DLD1 and DLD2 antibodies,the adhesion rate respectively decreased from 1%to 0.15%and 0.28%in the climbing plate,and decreased from25%to 7.5%and 12.5%in coelomocytes of A.japonicus,and the adhesion quantity of V.splendidus in various tissues of A.japonicus also decreased,which proved that dihydrolipoamide dehydrogenase participated in the adhesion function of V.splendidus to A.japonicus.The effect of DLD1 on adhesion is stronger than that of DLD2.（3）Through the construction of fliC overexpression vector p ET-28a GFPFli C,the fliC gene was successfully overexpressed in V.splendidus,and the fliC overexpression strain Vs/p ET-28a GFPFli C was obtained.The growth ability,swimming ability and biofilm formation ability of the overexpression strain were measured.The growth speed of fliC overexpression strain was stronger than that of wild type strain but there was no effect on the maximum biomass.Swimming ability and biofilm formation ability were stronger than that of wild type strain;By comparing the adhesion ability of fliC overexpression strain and wild type strain to the climbing plate,the coelomocytes and the individuals of A.japonicus,the adhesion rate of fliC over-expression strains to climbing plate increased from 1%to 5%and the adhesion rate of fliC over-expression strains to coelomocytes of A.japonicus increased from 25%to 39%.The amount of adhesion in intestinal tissue of A.japonicus reached 8.0×10⁶CFU/g,which was 2.5 times higher than that of wild type.These results proved that fliC gene was closely related to the adhesion ability of V.splendidus.The results showed that the main colonization sites of V.splendidus in A.japonicus were the respiratory tree,intestinal tissue and coelomic fluid.On the other hand,the adhesion function of V.splendidus is closely related to dihydrolipoamide dehydrogenase and flagellin.It is speculated that both dihydrolipoamide dehydrogenase and flagellin are the adhesion factors of V.splendidus,which lays a foundation for further study on the pathogenic mechanism of V.splendidus.