Development Of Chicken Ornithobacterium Rhinotracheale Oil Emulsion Inactivated Vaccine And Outer Membrane Protein Vaccine

Ornithobacterium rhinotracheale(ORT)is a respiratory infection that seriously endangers the chicken industry.ORT often harms broilers and turkeys aged 3-6 weeks,causing acute respiratory diseases.The aquaculture industry has caused large economic losses.The prevention and treatment of nasal tracheal bacillary disease mainly adopts two methods of adding antibiotics in feed or drinking water and immunization prevention by vaccine.Due to the drawbacks of antibiotic treatment foreign countries have developed,including live vaccines,inactivated vaccines and recombinant vaccines,for the prevention of nasal tracheal avian bacillosis.In this study,the oil emulsion inactivated vaccine was developed by using formaldehyde inactivated bacteria,and the protein gene was expressed by prokaryotic expression using genetic engineering technology,and the expression product was purified.The animal immune protection test was carried out to evaluate the immune antibody level and immune protection rate of the two vaccines.To lay the foundation for the prevention and treatment of nasal tracheal avian bacillosis.1.Study on the biological characteristics of ORTIn this study,the ORT isolates frozen in the laboratory were rejuvenated and identified.The results of Gram staining,biochemical identification,PCR identification and animal regression tests were similar to those reported in foreign countries,and serological tests showed The isolate is serotype A.Then the spectrophotometric method and plate colony counting method were used to determine the growth curve of A.faecalis,and the relationship between OD600nm and CFU of 16h Nasobacteria was studied.The results showed that the growth curve was basically the same,and the nasal trachea bird was obtained The logarithmic growth period of Bacillus is 2?16h,the stable period is 16?20h,and it enters the decay period after 20h,and the regression equation for OD600nm and CFU suitable for the logarithmic growth phase of A.faecalis is established:y=2.7591x-0.0897(R2=0.9973).Finally,the LD50 was determined and the LD50=1.19×1010CFU/mL was calculated according to the Karber's method,which provided a reference for the effective utilization and control of the growth of Nasobacteria.2.Development of inactivated ORT oil emulsion vaccineIn this study,glutaraldehyde was used to treat the polystyrene ELISA plate,and the whole cell antigen of A.sinensis was dried and coated.The optimal antigen coating concentration was determined by square matrix titration test to be 108 CFU/mL.It was 1:100 and it was verified by experiments that it has good specificity and repeatability.The isolated and identified A.faecalis was cultured to logarithmic growth phase and inactivated with 0.2%formaldehyde solution for 24 hours to prepare inactivated vaccine.The vaccine was tested for quality.The results showed that the vaccine had good emulsifying effect,aseptic growth and endotoxin content.In compliance with the requirements,there was no adverse reaction after vaccination of 7-day-old SPF broilers.They were vaccinated at 7 and 14 days of age,and their antibody levels were detected by indirect ELISA.The mice in the immunized group and the challenge group were challenged at 21 days of age to evaluate the immune effect and the ability to protect against infection.The results showed that the vaccines with different antigen contents after two vaccinations had better immune effects and could prevent the infection of Nasoche.3.Development of ORT outer membrane protein vaccineAfter reconstitution of the recombinant PGEX-OMA87 constructed and frozen in the laboratory,the OMA87 protein was expressed in a large amount and emulsified with Freund's adjuvant.The 7-day-old SPF chicken was used as the experimental animal to check the quality of the vaccine.The results showed that The vaccine has good emulsifying effect,aseptic growth,endotoxin content meets the requirements,and there is no adverse reaction after SPF broiler inoculation.The immune effect and the ability to protect the attack were evaluated.The results showed that the vaccines with different antigen contents after two vaccinations had better immune effects and could effectively prevent the infection of Nasobacteria.