Study On Safety And Efficacy Of Genotype Ⅱ Grass Carp Reovirus Oil Adjuvant Inactivated Vaccine

Grass carp hemorrhagic disease caused by grass carp reovirus（GCRV）has become one of the most prominent problems in freshwater aquaculture in China.Among them,genotype II GCRV is the most popular virus genotype which can cause serious clinical symptoms of grass carp.At present,some studies have confirmed that the inactivated vaccine for grass carp hemorrhagic disease is effective ingrass carp hemorrhagic disease.However,inactivated vaccines have problems such as large amount of antigen,short duration of immunity,and low immunity.Adjuvant is an immunopotentiator that can enhances non-specific immune responses and achieves ideal immune responsewith a small amount of antigens.In this study,the four oil adjuvants GR208（W/O/W）,Montanide^TMM ISA 763A VG,5 sticky white oil and 7 sticky white oil were emulsified with inactivated genotype II GRCR HuNan1307 at the ratio of 2:1 respectively.The stability,immune absorption effect,safety and immune efficacy of the adjuvant vaccine were evaluated and the most appropriate adjuvant vaccine for grass carp hemorrhagic disease was selected to provide important basis for the development of immune control products.The full text is divided into the following three parts:Study on the stability and abdominal absorption of GCRV oil adjuvant vaccineTake a amount of prepared adjuvant vaccine in a refrigerator at 4°C,and continue to observe whether the vaccine is layered.The adjuvant vaccine was injected intraperitoneally to immunize grass carp with 90 tails in each group.Five grass carps were observed anatomicly from each group at the 1st,2nd,3rd,4th,5th,8th,and 12th week after immunization.The results showed that the four groups of adjuvant vaccine groups did not find the phenomenon of virus separation and had a good stability.There was no adhesion between mesenteric membrane and abdominal wall in each experimental group.At 12th week,a certain amount of adjuvant vaccine residue was observed in each experimental group.Adjuvant vaccine absorption rate:GR208 vaccine group>5 sticky white oil vaccine group>7 sticky white oil vaccine group>Montanide^TMM ISA 763A VG vaccine group.Safety evaluation of GCRV oil adjuvant vaccine:This study evaluated the safety of adjuvant vaccines by counting the survival rate,growth performance data and examining the pathological lesions of visceral tissues.The results showed that after 3 days of immunization,all groups of grass carp showed different degrees of death.Among them,5 sticky white oil and 7 sticky white oil adjuvant groups had the lowest survival rates.After immunization with each group of vaccines,different degrees of pathological changes such as cell swelling and vacuolar degeneration were observed in grass carp liver tissues,but did not affect the life characteristics of grass carp.Among them,5 sticky white oil adjuvant vaccine had the most serious damage to liver tissues.After the immunization,the body weight and body length of grass carp had no significant difference with the negative control group（P≥0.05）,indicated that the oil adjuvant did not affect the growth performance of grass carp.Combined with liver injury,grass carp growth performance and stress condition,GR208 and Montanide^TMISA 763A VG have better safety.The efficacy evaluation of GCRV oil adjuvant vaccine:Grass carp samples were collected on the 1st,2nd,3rd,4th,5th,8th,and 12th week after immunization.Tissue sections of the spleen which is the main immune organs were observed and specific antibody levels of grass carp were detected.Attack protection experiment were carried out at the 6th week after immunization.The results showed that at4th week and 8th week after immunization,the area of melano-macrophage centres in the spleen of each adjuvant vaccine group was significantly higher than that of the negative control group（P<0.05）and the adjuvant-free vaccine group indicated that oil adjuvant could improve the vaccine presentation.From the first week to the 12th week after immunization,the specific serum antibody levels of grass carp in the adjuvant vaccine group were significantly higher than those in the control group（P<0.05）,and at the 8w and the 12th week,they were significantly higher than those in the adjuvant vaccine group（P<0.05）.The results showed that each adjuvant group could significantly improve the relative immune protection of the vaccine compared with the non-adjuvant vaccine group.At the 8th week and 12th week,the antibody levels in the adjuvant-vaccinated group were significantly higher than those non-adjuvant vaccine group（P<0.05）,and the GR208adjuvant vaccine group achieves the highest antibody levels.The results of the challenge protection experiment showed that compared with the non-adjuvant vaccine group,each adjuvant group could significantly improve the relative immunoprotective effect of the vaccine,in which GR208 and 5 sticky white oil adjuvant vaccine group had the highest relative immune protection rate,witch was 100%.Comprehensive analysis of the immune response of the spleen,antibody levels and protective effects,GR208 adjuvant has the best immune enhancement from the four candidate adjuvants,.This study comprehensively compared the stability of oil adjuvant vaccines,safety,immune enhancement and immune efficacy.The results showed that oil adjuvants have good safety in grass carp and can significantly increase the antibody level,protection rate and the protection period.The oil adjuvant vaccine prepared with GR208 adjuvant and inactivated GCRV HuNan1307 emulsified at 2:1 is the best.