Immunoprophylaxis Technology Of Main Bacterial Diseases Of Yellow-head Catfish,Pelteobagrus Fulvidraco

Yellow-head catfish,Pelteobagrus fulvidraco is an economically important freshwater fish in China.Which is widely distributed in Hubei、Hunan、Sichuan、Guangzhou provinces,etc.In recent years,alongside the increase of intensive aquaculture of this species,diseases are becoming more and more serious and have caused huge economic loss.This study mainly focused on immunoprophylaxis technology of two main bacterial diseases of yellow-head catfish.This research is divided into three sections.Section Ⅰ:Isolation and identification of pathogenic bacteria in yellow-head catfish,P.fulvidraco.In this study,two strains of pathogenic bacteria were isolated from liver and kidney of infected yellow-head catfish from different farms,identified by morphological,and biochemical analysis,gene sequence analysis,etc.Healthy yellow-head catfish were experimentally infected with the bacterial suspension.Thus,two strains of pathogenic bacteria named XJH06 and JZ096 were isolated from the liver and kidney of diseased yellow-head catfish.Identification by by morphological,physiological and biochemical analysis,gene sequence and phylogenetic analysis demonstrated that the bacterial pathogen XHJ06 is Aeromonas veronii and JZ096 is Edwardsiella ictaluri.The results of drug-sensitive tests showed that XHJ06 and JZ096 were both sensitive to flufenicol,enrofloxacin,etc.Section Ⅱ:Inactivated A.veronii or E.ictaluri vaccine stimulates protective immune responses in yellow-head catfish,P.fulvidraco.In this study,formalin-inactivated A.veronii（F-AV）or formalin-inactivated E.ictaluri（F-EI）respectively as immunogen,adjusting concentration to three different gradient of1.0 x 10⁸ cfu/m L,1.0 x 10⁷ cfu/m L,1.0 x 10⁶ cfu/m L,by intraperitoneal injection of immune health yellow-head catfish,respectively.Therefore,the yellow-head catfish were immunized with the formalin inactivated vaccines and the immunization efficacy were evaluated by measuring lysozyme activity,total protein,the serum C3 in serum,specific antibodies the m RNA expression of immune-related genes and the survival rate.The experimental results show that when the concentration of antigen of 1.0 x 10⁸ cfu/m L,the immune effect is significantly higher than the other group.In this immunization group,lysozyme activity,total serum protein,serum C3 increased significantly at 7 dpi,the serum antibody titer enhanced significantly at 21dpi.In the kidney head,the Ig M,MR and Ctl gene expression were significantly up-regulated.The challenge results showed that the RPS were 48.0%and 52.0%,respectively,against A.veronii or E.ictaluri infection.Section Ⅲ:A bivalent vaccine comprised of inactivated A.veronii and E.ictaluri stimulates protective immune responses and promoting effect of astragalus polysaccharide in yellow-head catfish,P.fulvidraco.A bivalent vaccine was generated,containing two formalin-inactivated bacteria at different mixture ratio in volume of 3:7,5:5 and 7:3 of A.veronii and E.ictaluri with a concentration at 1.0 x 10⁸ cfu m L^-1 for each.Therefore,the yellow-head catfish were immunized with the bivalent vaccines and the immunization efficacy were evaluated by measuring lysozyme activity,total protein,the serum C3 in serum,specific antibodies against A.veronii or E.ictaluri,the m RNA expression of immune-related genes and the survival rate against A.veronii or E.ictaluri,etc.The results showed that the bivalent vaccine with a mixture ratio of 3:7 of A.veronii and E.ictaluri presented the highest immunization efficacy.In this immunization group,the lysozyme activity,serum total protein and serum C3 had a noticeable improvement at 7 dpi,then decreased at 14 dpi and returned to normal levels at 28 dpi.Immunoglobulin M（Ig M）gene was significantly up-regulated to the peak at 7 dpi in the spleen and at 14 dpi in the kidney after immunization,then gradually declined to intital level.Mannose receptor（MR）and C-type lectin（Ctl）genes significantly increased and reached their maximum at 24 h after immunization both in spleen and head kidney.Higher antibody levels were observed in vaccinated fish at day28 post-immunization.The challenge test showed that the relative survival percent in the immunized group with the mixture ratio of 3:7 of A.veronii and E.ictaluri was 49.1%and59.1%against A.veronii and E.ictaluri infection,respectively.And then,containing two formalin-inactivated bacteria at different mixture ratio in volume of 3:7 f A.veronii and E.ictaluri with a concentration at 1.0 x 10⁸ cfu m L^-1 fo as immunogen,add astragalus polysaccharides as adjuvant,respectively with adding astragalus polysaccharides adjuvants,no adjuvant group and PBS group to intraperitoneal injection of healthy yellow-head catfish.Therefore,the yellow-head catfish immunization efficacies were evaluated by measuring lysozyme activity,total protein,the serum C3 in serum,specific antibodies against A.veronii or E.ictaluri,the m RNA expression of immune-related genes and the survival rate against A.veronii or E.ictaluri,etc.The results showed that APS adjuvant has obvious synergistic effect on the bivalent inactivated vaccine of A.veronii or E.ictaluri infection.The lysozyme activity,serum total protein and complement C3 peaked at 14 dpi after immunization.Serum antibody titers in both immunization groups reached the maximum at 28 dpi.The expression of Ig M gene was up-regulated in the head kidney and reached the peak at 14dpi,and in the spleen and reached the peak at 7 dpi.The MR gene and Ctl gene in the head kidney and spleen were up-regulated to the maximum at 24h,and then decreased slowly but were always higher than that in the non-adjuvant group and the control group.The challenge results showed that the RPS of the APS vaccine group were70.0%and 79.1%,respectively,against A.veronii or E.ictaluri infection.