Primary Research On The Development Of Mycoplasma Synoviae Inactivated Vaccine

Avian mycoplasmosis(AM)is a chronic subclinical upper respiratory disease cuased by Mycoplasma synoviae(MS).Infections in avian show air sacculitis,tenosynovitis,joint and foot pad swelling accompanied inflammatory suppurative effusion,which results in great economic loss in poultry industry.So far,commercialized vaccine of MS is unavailable in poultry,The aim of this study is to developed an inactivated vaccines with good clinical protection to control the infection of MS.To make inactivated vaccine with good clinical protection,a virulent strain of MS named CHN-J2-2016 was isolated from a poultry farm in Binzhou city of Shandong province.After three generation,the MS was identified by PCR using 16S rRNA sequence and analyzed the gene of isolated strain with BLAST program.The results showed that the shape of the isolates was typical"poached egg"under the microscope,A specific band was amplified from the isolates using PCR assay with 16SrRNA primers.The homologies of 16S rRNA nucleotide sequences were 99%among CHN-SQ27-2017 strain and MS standard strain(WVU1853).The results demonstrated that the isolated of CHN-J2-2016 strain was mycoplasma synoviae.Then 21-day old chichens were infected with CHN-J2-2016 and the chickens were randomly selected from each group at 11 dpi and 21 dpi to detect the pathology.Results of the pathology showed that the CHN-J2-2016 strain cuases foot pad lesions and obvious synovitis,which suggested that the isolated of CHN-J2-2016 strain had high pathogenicity and can be used as a candidate of inactivated vaccine.In order to detect the growth characteristics of the CHN-J2-2016 strain,and determine the harvest time of antigen.The color change units(CCU),colony forming units(CFU),protein concentration and nucleic acid levels at 108 h of the CHN-J2-2016were determined.Reaults of the CCU and CFU showed that the MS reachs in the logarithmic phase at 12 h~48 h under the modified Frey culture medium,and the number of the bacteria reached the peak at 48 h with 10~8 CCU/mL as determined by CCU.Moreover,the viable count was up to 1.53×10~8 CFU/ml at 48~54 h and the protein concentration of MS increased rapidly from 6 h to 48 h,with a peak at 48 h(390.8ug/mL).The nucleic acid level of MS increased rapidly from 12 h to 48 h,which reached at 20.56.Collectively,the best time for antigen harvest in vaccine production process is 48~54 h.Four vaccines has good physical property and stored at 4°C for a half year.To study the immunogenicity of the inactivated vaccines,190 chickens were choosed and were randomly divided into 19 groups.All chickens were immunized firstly at 30 days,and strengthened immunized at 60 days.The first nine groups were immunized with four different vaccine combined with four different adjuvant through subcutaneous injection.Then the serum of each group was detected by ELISA after the first immunity from 7 d to 180 d.The following 10 groups were immunized again with the aboved vaccines through subcutaneous injection.At 42 days after first immunized,5 chickens were selected to determine the pathology.Results showed that the serum antibody titers of each immunized groups were slightly increased and only the 71R VG+Ag group showed significantly increasing after the first immunity,then the antibody titer declined thereafter.Results of animal tests showed that all the immuned groups displayed different symptoms and MS can be isolated from the throat of cotton swabs.After the detection of DNA genome extracted from tracheal tissue,the circulation value of immune group was lower than control group and the tracheal microscopy showed that all groups had a thickened tracheal mucosa at different levels,but the tracheal injury of vaccine group were weaker than adjuvant group.Patholog showed that the air sac has no significant differences among the groups,but the foot pads and joints showed different degrees of swelling with suppuration.Consequently,the results showed that all of the vaccines can induce the body to produce a antibody response,but only the 71R VG+Ag group can stimulate the body to produce high levels of antibodies and had a good clinical protective effect.In this present study,we have isolated and identified one strain of Mycoplasma synoviae CHN-SQ27-2017 as a vaccine candidate strain.We determined that the best harvest time of antigen is at 48 h and prepared four vaccines.Animal tests showed that four vaccines can stimulate the body to produce antibody and the 71R VG+Ag group showed the highest of clinical protective.