Development And Identification Of An Attenuated Mycoplasma Hyopneumoniae Aerosol Vaccine Produced By The Ultrasonic Method

Mycoplasma hyopneumoniae(Mhp)is a major causative agent of Mycoplasma pneumonia of swine(MPS),it can cause swine chronic respiratory diseases.It has been confirmed that Mhp live vaccine is effective by pulmonary injection,but this method have some difficulties for clinical immunization.In this study,aerosol immunization techniques was used in Mhp live vaccine,on the one hand to solve the technical drawbacks of Mhp vaccine immunization by pulmonary injection,on the other hand to provide a new automated immunization method for swine.In this study,Mhp vaccine was produced by ultrasonic nebulization,and the dilution type of vaccine and the formulation of protection were screened respectively.In terms of screening dilutions,measured resistance atomization and instant vitality through collected vaccine solution before atomization and the atomized.Seven kinds of diluents including the deionized water,PBS,0.9%NaCl,10mmol/L Tris-HC1,50 mmol/L Tris-HC1,100 mmol/L Tris-HC1,and 200 mmol/L Tris-HCl were optimized.and PBS was the most appropriate diluent.Based on the optimal diluent,we use CCU50 and real-time PCR methods to screen the protection agent in 4.47%trehalose,0.5%gelatin,0.1%dextran and 5%glycerol.The result showed that the 0.5%gelatin was selected to be used as the protection agent.In this study,Mhp vaccine aerosols by ultrasonic atomization were identified in morphology.The Topas Lap322 particle spectrometer was applied to measure the particle size firstly,we found that the particle size is relatively big when trehalose or glycerol was used as a protective agent.Using gelatin as a protective agent,the particle size is relatively small,while on the whole aerosol particle size by ultrasonic nebulizer between 2-4?m is basically in line with the lower respiratory tract of swine.Secondly,Then in order to observe the characters of Mhpvaccine aerosols,we use cover glass to select the aerosol vaccine,then the shape,size and uniformity of which were observed with confocal microscopy.The results revealed that the percentage of Mhp in ultrasonic nebulizer aerosol vaccine stock solution and the spray vaccine solution diluted 10-fold were respectively 76.52%and 58.82%.A single aerosol contains an average of 3.06 and 1.51 Mhp respectively,moreover the atomized aerosol were uniform and stable.In this study,the diluent and the protection agent of Mhp live vaccine was screened,and the particle size was measured.With the addition of the observation,it can be inferred that the Mhp live vaccine aerosols by ultrasonic atomization can be used in the further study.