A Preliminary Study On The Whole Vaccine Of Pseudomonas Plecoglossicida

The large yellow croaker,Larimichthys crocea,was one of major marine economic fish in China,more and more diseases plagued the aquaculture and hindered the healthy development of large yellow croaker in recent years.Among them,the toxicity of "White Spot" Disease in large yellow croaker is extremely serious for intensive farming,causing huge losses to farmers.In this study,we focused on Pseudomonas plecoglossicida which separated and identified from large yellow croaker,and conducted a preliminary study on the whole vaccine of Pseudomonas plecoglossicida.The main contents are as follows:1.Establishment of experimental animal model of Pseudomonas plecoglossicida infection under laboratory conditions.We selected several commonly experimental fish and the test fish were challenged by soaking,intraperitoneal injection and intramuscular injection.We Screened out fish that are susceptible to Pseudomonas pseudomonas and have typical symptoms of "White Spot" symptoms as a laboratory pathology model organism.The result showed that Epinephelus coioides can be used as a pathological model of Pseudomonas plecoglossicida in laboratory which was more sensitive with high rate of morbidity and "White Spot".2.Inactivation conditions screening of Pseudomonas plecoglossicidaThe inactivation method of bacterial liquid contain physical and chemical inactivation.In order to obtain the best inactivation method of Pseudomonas pseudomonas,chemical inactivation was more extensive in bacterial and viral applications.In the present study,methanal,phenol and chloroform were introduced to inactivate Pseudomonas plecoglossicida under different concentration and temperature of the inactivation agent.Three kinds of inactivated fluid were selected for immunity test of Epinephelus coioides,which prepared under the condition of moderate inactivation agent.Then,we can find out a kind of inactivation scheme for the protection of the antigen with good immunogenicity.The result showed: there were three inactivation schemes with mild drug that 28? 12 h for 0.2% methanal,28?24 h for 0.5%phenol,28?12 h for 3.5% chloroform,respectively.We find the inactivated vaccine prepared by methanol had best immune effect after Epinephelus coioides immunization test of three Pseudomonas plecoglossicida vaccine prepared by inactivation.3.Inactivated vaccine adjuvant screening of Pseudomonas plecoglossicidaWe Added different adjuvants to enhance the immune effect of Pseudomonasplecoglossicida for the screening of immune adjuvants suitable of Pseudomonas plecoglossicida.We choose Freund's adjuvant,white oil adjuvant,alhydrogel,206 adjuvant and propolis adjuvant for preparation of adjuvant vaccine with Pseudomonas plecoglossicida vaccine.The result showed: Compared to control,the immune protection rate of Freund's adjuvant,white oil adjuvant,alhydrogel,206 adjuvant and propolis adjuvant for Epinephelus coioides were 91.7%,83.33%,80.56%,72.22% and 66.67%,individually.These indicated that the adjuvants can enhance the immunogenicity of antigens and improve immune function.4.Preparation technology of oral microsphere vaccineWe used alginate as the wall material and buried the Pseudomonas plecoglossicida vaccine.After single factor test,we find the condition of dry spray preparation was that: the concentration of sodium alginate was 2.5%;the inlet temperature was 140?;sampling rate was 600 mL/h;atomization pressure was 0.2 Mpa.It has good acid resistance and good release performance in intestinal solution,because of smaller particle size of microsphere vaccine.5.Evaluation of Immune Effect of Pseudomonas plecoglossicida Oral Microcapsule Vaccine on Epinephelus coioidesIn order to evaluate the immunological effect of sodium alginate-encapsulated Pseudomonas plecoglossicida oral microcapsule vaccine,which was prepared according to the established preparation process,and oral administration of Epinephelus coioides was carried out.The immune indexes of superoxide dismutase(SOD),lysozyme and serum agglutination antibody titers were measured,and the immune protection effect of the vaccine on Epinephelus coioides was evaluated by artificial infection test.The results showed that the activities of superoxide dismutase and lysozyme in Epinepheluscoioides were significantly improved after the microspheres were immunized with Epinephelus coioides.And the peak value of serum antibody titer was 1:16 and the relative immunoprotection was 28.94%,which was significantly higher than that of other experimental groups.These indicated that sodium alginate-encapsulated Pseudomonas plecoglossicida microspheres had a certain protective effect on Pseudomonas plecoglossicida,and the microspheres prepared by sodium alginate had a protective effect on the antigens.6.Protective effect of attenuated live vaccine of Pseudomonas plecoglossicida on Epinephelus coioidesWe constructed stable silencing strains of Pseudomonas plecoglossicida by gene silencing.The results showed that the mortality of Epinephelus coioides was significantly decreased.However,silent strains also have some toxicity,which fail to meet the requirements of attenuated vaccine.