Evaluation Of The Safety Of Mycoplasma Hyopneumoniae Aerosol Vaccine By 3D Cell Culture Technology

Mycoplasma hyopneumoniae(Nhp)is the pathogen of mycoplasmal pneumonia of swine(MPS),a chronic respiratory disease with high morbidity and low mortality.The disease can cause secondary infection.Up to now,our country uses widespreadly the inactivated vaccine to prevent and control MPS.At present,M.hyopneumoniae 168 attenuated strain vaccine made by institute of veterinary medicine,Jiangsu academy of agricultural sciences in 2007 has been widely used for industrial production,but it can only be immunized by intrapulmonic route.Furthermore,intrapulmonic injection for the attenuated vaccine is labor intensive and time consuming.Aerosol vaccination presents a needle free,high throughput,and efficient platform for vaccine delivery,and has been widely applied in poultry vaccination.The first attenuated M.hyopneumoniae aerosol vaccine was prepared and demonstrated to penetrate and adhere to the lower respiratory tract of piglets successfully in 2013.However,the safety and immunized doses of aerosol vaccine remain to be further studied.At present,it is widespread that the toxicity of aerosol nanoparticles was evaluated using an in vitro air-liquid interface(ALI)exposure system where cells are cultured on thin porous membranes in cell culture inserts with cell culture medium only on the basal side of the insert.The advantage is to provide an in vitro exposure setting that is more representative of pulmonary exposure.Compared to submerged exposure systems,ALI methods permit the particles to maintain their intrinsic characteristics until they reach the cells,thereby allowing for a better control of the effective dose.Therefore,this study was aimed to evaluate the safety of M.hyopneumoniae aerosol vaccine using in vitro exposure systems by an ALI.Firstly,this study was aimed to establish an air-liquid interface(ALI)cultivation model of continuous swine tracheal epithelial cells(STEC)cell line by optimizing the cultured time,serum concentration and adding factors of cell culture media,The results showed that the best microvillus on the surface of STEC cells was observed at the tenth day and cultured in cell culture medium with 10%FBS and 15 ng/mL retinoic acid.Therefore,we carried out the safety evaluation test of M.hyopneumoniae aerosol vaccine by cultivating the cells for 10 day.The experiment was divided into the following two parts:1 The pathogenic mechanism of mycoplasma hyopneumoniae strains with different virulence on 3D cultured swine tracheal epithelial cells,An air-liquid interface(ALI)cultivation model of continuous STEC cell line,closer to the status in vivo,was developed for Mhp infection.Mhp strains with different virulence(virulence strains NJ,intermediate virulence strains AH,and attenuated strains 168L)were selected for respective infection with the same dose.The integrity of cell monolayer and the vitality of the infected cells were respectively evaluated by TEER value,Alamar Blue assay and lactate dehydrogenase(LDH)release assay.The ability of the differentiated cells to secrete MUC5B mucin against Mhp infection was measured by laser confocal and fluorescence spectrophotometer.To further study the pathogenic mechanism of Mhp infection,the cellular oxidation system of the infected STEC cells,including the secretion of NO and endogenous ROS were detected,respectively.The effect of NAC antioxidant on cell damage of mycoplasma infection group was observed and was further demonstrated.The results showed as follows:The infection of virulence strains NJ and intermediate virulence strains AH but not attenuated strains 168L could significantly roughen,disorder or damage the microvillus and cilia on the surface of STEC cells.The resistance of cell monolayer was decreased after infection,which was positively correlated with the virulence of Mhp strains.Mhp strains NJ and AH could significantly reduce the vitality of the infected cells using Alamar Blue and LDH release assays,especially at 48 h post-infection.Mhp strains could also promote the differentiated cells to secrete MUC5B mucus against Mhp infection,and the stronger the virulence of the strain was,the more mucous was secreted.It was found that except the 168L strain,AH strain and NJ strain could both significantly stimulate STEC cells to secrete NO and endogenous ROS,which could cause the oxidative stress reaction of cells significantly.After the anti-oxidation treatment of NAC,the ROS and mucus secretion of virulence strains NJ,intermediate virulence strains AH and the attenuated strains 168L groups decreased significantly,while the cell activity and resistance value increased significantly compared with the anti-oxidation untreated group.2 Evaluation of the safety of aerosol inoculation method by 3D cell culture technology.In this study,the aerosol sprayer device for the evaluation of mycoplasma hyopneumoniae aerosol vaccine was established and optimized using in vitro exposure systems by an ALI.An air-liquid interface(ALI)cultivation model of continuous STEC cell line,closer to the status in vivo,was developed for aerosol infection,Mhp attenuated strain 168L by aerosol and droplet methods were selected for respective infection with the same dose.The integrity of cell monolayer and the vitality of the infected cells were respectively evaluated by TEER value,Alamar Blue assay and lactate dehydrogenase(LDH)release assay at 3 h and 48 h post-inoculation(pi).The ability of the differentiated cells to secrete MUC5B mucin against aerosol infection was measured by laser confocal and fluorescence spectrophotometer at 3 h pi and 48 h pi after infection.To further study the pathogenic mechanism of aerosol infection,the cellular oxidation system of the infected STEC cells,including the secretion of endogenous ROS and proinflammatory factor(IL-6、TNF-α and IL-1β)were detected respectively at 3 h pi and 48 h pi.Ultimately,the safety evaluation of M.hyopneumoniae aerosol vaccine was carried out,and the results are as follows:At 3 h pi and 48 h pi,the microvillus and cilia on the surface of STEC cells infected by Mhp attenuated strain 168L with droplet method was consistly not significantly different from the control group.However,the infection of Mhp attenuated strain 168L by aerosol method could significantly roughen,disorder or damage the microvillus and cilia on the surface of STEC cells at 3 h pi,and as the incubation time was extended to 48 h pi,the growth status of the microvillus and cilia gradually recovered.At 3 h pi and 48 h pi,the resistance of cell monolayer and cell vitality of STEC infected by Mhp attenuated strain 168L with droplet method was consistly not significantly different from the control group.However,the infection of Mhp attenuated strain 168L by aerosol method significantly reduced the vitality and resistance of cell monolayer of the infected cells using Alamar Blue,LDH release and TEER value assay,and as the incubation time was extended to 48 h pi,the vitality and resistance of cell monolayer of the infected cells gradually recovered.The infection of Mhp attenuated strain 168L by aerosol method could also promote the differentiated cells to secrete MUC5B mucus,and as the duration of infection increased,the secretion of mucus decreased.The variation trend of cellular secretion of endogenous ROS and proinflammatory factor(IL-6、TNF-α and IL-1β)after the infection of Mhp attenuated strain 168L by aerosol method was consistent with its corresponding cell vitality,resistance and function of secreting MUC5B mucus.It was indicated that the pathogenic mechanism of the aerosol infection of Mycoplasma pneumoniae was closely related to oxidative stress and inflammatory factor secretion.