Effect Of Saponins On The Presentation Of Antigens In The Mucosal Tissue Of Turbot

Bacteriosis are the most serious diseases in aquaculture at present.More thantwenty kinds of pathogens that could cause huge economic losses to the fishaquaculture were identified in the world.Thus,prophylaxis and control of fish bacterialdisease by vaccine has practical significance.In the past decade,fish disease controlwere largely depend on using antibiotics.But today,the effect of using antibiotics isbecoming serious.More pathogenic bacteria obtained drug resistance,immunity of fishagainst disease were lower than that in the wild,drug residues in aquatic products andwater could cause environment pollution.Food safety and human health are seriouslyaffected by using antibiotics.Application of vaccine in the prevention of fish bacterialdiseases had achieved good results.Among the modes of fish vaccinations,immersionis the preferred method of immunization for its simple operation and safety.Butvaccination of fish by direct immersion cannot enhance the uptake of antigen throughthe epithelial skin and gills.Thus,the research and develop of high efficiency and lowtoxicity adjuvant to increase the effectiveness of immersion vaccine have become amain task an home and abroad.In this study,the research team had screened saponin asadjuvant,combined with Vibrio anguillarum vaccine immunization turbot.Researchsaponin in turbot antigen absorption intensity before and after the surface,changeantigen in areas of mucosal tissue by time,explore the role of saponins promotingmucosal tissue absorption antigens.In this study, four parts of research contents were included.Part I：Fluorescein isothiocyanate (FITC) was used as the marker to mark severalpathogenic bacteria and probiotics in seawater,and optimize the labelingcondition.The results show that the optimal concentration of FITC was in the range of20to100g/mL and the optimal time was in the range of60to120min.The fluorescent signal from V.anguillarum labeled FITC was more stable when it wasstored at-20℃and-70℃than those that stored at4℃and25℃.The stability offluorescent signal from formaldehyde inactivated V.anguillarum labeled FITC wasbetter than that from the control,and no difference was found in the quenching effectin different intensity of natural lighting.After3times of freezing and thawing,thefluorescent signal from V.anguillarum labeled FITC frozen at-70℃was morestable.The results above provided useful technical parameters when FITC was used asa marker to label the marine bacteria.Part II： The experiment has to mark V.anguillarum immunization turbotcombined with saponin,detect fluorescence intensity in gills and different parts of thebody surface as the main mucosa.The results show that saponins could enhanceantigen presentation.Gills epidermis has enriched fluorophores.With the increase ofconcentration of saponins and immersion time,the number of fluorophores andfluorescent intensity gradually increased.Detect the different parts of body surfaceskin,the skin on the back of fluorescence intensity is higher than the ventral,and on theback of the gill edge skin and lateral line of fluorescence intensity is higher than thatof any skin obviously.The fluorescence signal is present in the epidermis,whichindicates that gill edge and lateral line skin are two important antigen presentingparts,these antigen-presenting cells exist in the epidermal layer of the organization.Part III：On the basis of the second part,with the fluorescent labeled bacteria andindirect immunofluorescence methods for detecting the intensity of antigenabsorption,the change of antigen in the remaining time in areas of mucosal tissue.Theresults show that the two methods complement each other and improve theaccuracy.With the immersion time extending,mucosal tissue fluorescence intensityincreased including gill and gill edge skin and lateral line skin,and the highconcentration (40mg/L) fluorescence intensity is stronger than the low concentration(20mg/L) with the saponin immersion.The results indicate that saponin as adjuvantscould enhance mucosal immunity and open a new research direction and a newtechnical reference for V.anguillarum and other pathogens. Part IV：Using the methods of tissue sections and scanning electron microscopyto further explore the mechanism of antigen-presenting.Tissue sections could maintainstructure integrity and show a positive signal in a specific position clearly.Thescanning electron microscopy can detect the gathered bacteria and observe changes inthe structure of the skin surface.The above experimental results showed that saponinshave enhanced the role of antigen presentation.Its presented site is surface cells in gilland gill edge skin and lateral line skin.Explain the mechanism of saponin from thehistological level.