Exploration On The Manufacturing Technology Of Mycoplasma Synoviae Inactivated Vaccine(HN01 Strain)

Mycoplasma synoviae(MS)infects chickens and turkeys and mainly proliferates in synovial sac and tendon sheath.The infection usually shows a slow progress and causes joint enlargement,exudative synovitis,mucocystitis or tenosynovitis.The disease happened in caged chickens is hardly detected and eradicated.Co-infected of MS with other pathogens in poultry is popular in clinical,and increases the mortality and economic loss as well.The killed and attenuated vaccines have been respectively commercialized in other countries.Here,we also developed an inactivated vaccine of Mycoplasma synoviae,named HN01 strain.This study focused on the manufacturing technics of this vaccine,including culture medium screening,antigen fermentation,concentration and inactivation techniques.1 The aim of this study was to select a low-cost,easily-formulated and efficient culture medium.The physicochemical index and culture performance of four kinds of culture mediums,modified MS medium,modified Frey's medium,KM2 medium and modified KM2 medium,were compared using the color change unit(CCU)assay.The results showed that the modified MS medium had a fewer pH value change after sterilization and a higher titer(108?9 CCU/ml)than other mediums.The modified MS medium could be the candidate for cultivation of MS inactivated vaccine(HN01 strain).2 The freeze-dried Mycoplasma synovial is(HN01 strain)was revived with the improved MS liquid mediums.Then Mycoplasma synovialis were inoculated in the same medium in a ratio of 2%,5%,10%,15%and 20%,respectively.The optimum inoculation amount was screened by the results of Pure Test Method and CCU assay.Using 5L fermentor,dissolved oxygen was automatically adjusted to 10%,20%,40%and 80%respectively.The optimum dissolved oxygen was selected according to the results of Pure Test Method and CCU assay.Mycoplasma synovialis were inoculated into 5L fermentor and cultured for 12h,16h,20h,24h,28h and 32h respectively.In order to make the optimal fermenter culture curve,the cultures were taken aseptically and then the pH of the culture was recorded,Pure Test Method and CCU assay were performed.In order to figure out the optimum ratio of fluid replenishment and the optimum times of fluid replenishment,0.1%,0.2%and 0.5%arginine were added into the culture with different times during the stable growth period.These results will provide important parameters for mass production.Our studies show that 10%of inoculation ratio,80rpm of stir speed and 20%of the dissolved oxygen could prolonged the growth stable stage of HN01 strain to 13?19 hours by continuous culture technology.The titer of live bacteria of MS(HN01 strain)could reach 109.0CCU/ml stably.3 In this stage,the titer of Mycoplasma synovialis(HN01 strain)was concentrated by using ultrafiltration with a 100 K molecular weight cut-off membrane and 16 000 r/min continuous centrifuge.Both of the two methods could improve the concentration of Mycoplasma synovialis by 10 times.The harvested antigens were used for vaccine development to evaluate the feasibility of two concentrate methods.According to the physical and chemical properties,safety and efficacy of the test vaccines,both of the concentration methods,ultrafiltration and continuous centrifugal sedimentation,were proved for good propagation of HN01 strain.Both of the concentration methods are safe and effective.The physical properties of MS vaccine are uniform and stable without significant difference.Both of them are suitable for large-scale production and application of vaccine production enterprises.4 The MS antigen and concentrated MS antigen were put in sterile containers and both of them were divided into three groups.10%formaldehyde solution(prepared with sterilized saline)with a ratio of 0.1%,0.3%and 0.5%were added into the three groups respectively for two antigens.After fully mixing,the two kinds of antigen were inactivated at 37? for 12 hours,16 hours 20 hours,24 hours,30 hours and 36 hours,respectively.Then the inactivation test was carried out.The results showed that the concentrated MS antigen could be inactivated completely with 0.3%of formaldehyde solution at 37? for 16 hours.In order to guarantee the antigen has been completely inactivated,the inactivation process for MS was set as follows:adding formaldehyde solution to the antigen(the terminal concentration of formaldehyde was 0.3%according to the volume of the antigen),keeping incubation at 37? for 24 hours to inactivate,shaking or stirring every 2 hours.