Development Of Oil Emulsion Inactivated Vaccine Based On The Large-scale Culture Of FAdV4 With Torrent Bioreactor

Avian adenovirus(Fowl Adenovirus,fadv)is a common infectious disease pathogen in poultry and wild poultry in the world,divided into 3 subgroups of 5 species(A-E),12 serotypes,of which the serum type 4 is mainly manifested as pericardial effusion in clinical practice,the first to see the spread of the disease in 1985,The outbreak of the disease at a broiler farm in the Ankara area near Karachi,Pakistan,in March 1987 was characterized by pericardial effusion,from which Ankara's disease was named.Since 2013,the outbreak of serum Type 4 avian adenovirus in Shandong,Jiangsu,Jiangxi,Hunan,Yungui and the three eastern provinces has caused serious economic losses to the poultry industry.Foreign vaccines for the prevention of avian adenovirus have been developed abroad,but there is no vaccine or drug available to prevent serum type 4 avian adenovirus in China,which also brings considerable difficulty to the prevention and control of the epidemic,and the market is in urgent need of a dominant vaccine that can prevent the epidemic of avian adenovirus in the current domestic chicken herd.Subsequently,the domestic study of its pathogenic FAdV also carried out rapidly.At present,cell suspension culture technology has been successfully used in the production of foot-and-mouth disease,blue ear disease and avian influenza vaccine at home and abroad,compared with the traditional bottle wall culture,because of the continuous closed system,the operation steps are simplified,the opportunity of pollution is significantly reduced,the automatic control and monitoring of various production parameters is more convenient,It is of far-reaching significance to improve the cell density,increase the amount of toxicity,reduce the cost,reduce the pollution and further improve the production process of the avian adenovirus vaccine by changing the lengthy and cumbersome procedures of the previous single layer culture.1.Preparation of inactivated vaccine of Fowl adenovirus serotype 4,FAdV4In this study,FAdV4 was used as the research object.To improve the technics for producing the FAdV4 vaccine,the procedures for large-scale production of the FAdV4 were carried out in this study.With disposable cell culture bag,paper carrier,and serum-free medium,the paper carrier-based cell culture technology to propagate the FAdV4 was successfully established in the torrent bioreactor.Then five batches of FAdV4oil emulsion inactivated vaccine(JH strain)(SY-201601?SY-201602?SY-201603?SY-201604 and SY,201605)were produced,and the inspection results showed that these vaccine products met the quality testing requirements including physical properties and sterility.In addition,three batches of selected vaccine(SY-201602?SY-201603 and SY-201604)also exhibited excellent safety after the safety experimentson SPF chickens.Control parameters of cell suspension culture in AP20 bioreactor:medium serum dosage is 10%,temperature(T)is 37?,do is not less than 50%,pH is 7.0-7.2,speed is 40 rpm.The total number of initial cells inoculated with 200 g vector was(1±0.2)×109 cells,which were cultured for 6 days.The optimal exposure ratio of the virus in bioreactor was 0.1 MOI and 2%FBS was added as maintenance solution.The optimal harvest time was 94-98 hours after exposure,and the titer was 109.0tcid50/ml.2.Safety and immune efficacy of inactivated vaccine of Fowl adenovirus serotybe 4,FAdV4In order to study the protective effect of the vaccine on commercial chickens,three batches of selected vaccine(SY-201602?SY-201603 and SY-201604)were used on 14 day old yellow feathered broilers,white feather broilers and laying hens,ten chickens every goup.At the same time,5 chickens of the same age and the same breed were given the same amount of PBS as the control group.The neutralizing antibody titer was measured 21 days after immunization.At the same time,carry out drug protection test.After 14 days,the incidence of each group was observed.The results showed that the three batches of vaccines produced in the laboratory also had good immune protection for commercial chickens.Here,we used paper carrier to culture LMH cells at high density in automationcontrol bioreactor and explore the optimum conditions for viral culture and harvest,which could provide a theoretical basis and practical experience for the industrialized mass production of FadV4 vaccine.3.Fowl adenovirus serotybe 4,FAdV4 inactivated vaccine(JH strain)smallest target animals,dose safety test efficacy test for various pathwaysSelected 14-year-old SPF chicken 80,divided into eight groups,of which 1-4 groups using the lower neck,5-8 groups using leg chicken injection 0.3mL/only,respectively,three batches of vaccines and PBS control,14 days to observe the chicken's mental state,appetite and other changes,14 days after weighing and caesarean section.The results showed that there were no significant abnormalities in the mental state and appetite of the chickens in the experimental and control groups within 14 days,and after 14 days the weighing showed no significant difference between the subcutaneous neck and the intramuscular injection and control group,and the caesarean section showed that there were no abnormalities in all the immune sites of the chickens,indicating that the vaccine was well absorbed.The results of a single dose of vaccination showed that the vaccine was safe to inoculate 14-year-old SPF chicken in a single dose of subcutaneous and leg muscle injections.Select 14-year-old healthy non-yellow feather chicken,white feather broiler chicken,egg chicken each 15,of which 10 neck underarm immunization 201604 batches,and five injectionPBS as a control,All chickens were collected blood 21 days after immunity,and tested for anti-venom protection,neutralizing antibody efficacy after 14 consecutive days of observation.Each group of chickens poisoned within 14 days,2 times a day to observe,collect each group of dead chickens and section,observe organ lesions,14 days later,kill all the surviving chickens and section,observe the lesions of the organs.The results showed that 3 kinds of commodity chicken immune vaccine for 21 days,antibody test results showed that the immune group and antibody efficacy geometric average is 1:75.9,1:89.8 and 1:85.3,respectively,while the control group is not higher than 1:2,the survival rate statistics show that the control group were 100%sick within 14 days after the attack.The immune group was 100%protected.The results of the caesarean section showed that the disease of chicken within 14 days after the control group was poisoned showed that the disease was found in the avian adenovirus lesions,and the immune group caesarean section did not see any related avian adenovirus lesions.The results show that the vaccine has a good immune protection effect on commercial chickens.In this study,the high density culture of LMH cells was carried out through paper carriers,the best culture conditions and methods of FAdV4 culture and toxicity were explored in the automatically controlled bioreactor,and five batches of vaccines were tested,showing good safety and protection.The reforest provides theoretical basis and practical experience for the large-scale cultivation of FAdV4 and the preparation of high-efficiency inactivated vaccine,which is very practical.