Study On The Growth Curve And Inactivated Vaccine Of Mycoplasma Ovipneumoniae

Sheep mycoplasma pneumonia,caused by Mycoplasma ovipneumoniae,could infect sheep and goats resulting in atypical pneumonia and has wide distribution all over the world.The aim of this study was to develop inactivated vaccine of Mycoplasma ovipneumoniae.Nasal swabs and tissue samples of trachea and lungs of the sheep farms the clinical symptoms of pneumonia were collected in the slaughterhouse.Mycoplasmaovipneumoniae strains were isolated and cultured from samples and swabs using modified medium prepared in our laboratory.The strains were passaged and the growth curves were measured using suspensions diluted with the ratios of 1:6,1:10,1:20,1:30,1:40,1:50,1:100,1:200.Colony forming unit(CFU)and color change unit(CCU)were measured every 6 h after incubation and data were collected and transferred into curves.The results showed that the best passage ratio of mycoplasma pneumoniae in the laboratory was 1:6 or 1:20,the highest bacterial rate was 1.3 × 109 CFU/mL,and the best harvest time was 120h after inoculation.The cultures with culture titers more than 5×108 CFU/mL after being passaged with the dilution ratio of 1:200 were washed to a 109 CFU/mL growth titer.Aluminum hydroxide and 1%levamisole were used as separate adjuvants.Two kinds of inactivated vaccines were injected into 10 healthy sheep to test the biological security.After the safety test,swabs of 200 sheep were collected to isolate and identify Mycoplasma ovipneumoniae strains.The 200 sheep were divided into two groups and immunized twice with two kinds of inactivated vaccines separately via subcutaneous injection of neck.The first immunization was conducted on the first day and the second immunization was conducted 14 days later.The blood samples were collected after the sheep were immunized each time,and the serum was isolated by centrifugation to detect antibody level via indirect ELISA.The results showed that before immunization,the positive rate of strain isolation was 40%,and the positive rate of PCR was 45%;after the first immunization,the positive rate of strain isolation was 30%,and the positive rate of PCR was 35%;after the second immunization,the positive rate of strain isolation was 15%,and the positive rate of PCR was 15%.Compared with the control group,the positive rate of PCR and the positive rate of PCR were significantly decreased.The results of ELISA showed that when the P/N value was higher than 2.112,the antibody level in the serum of the immunized sheep was significantly higher than that in the control group.The antibody titer of sheep in the group of vaccine with aluminum adjuvant reached up to 1:256 and 1:1024 seperately after the first and second immunization.The antibody titer in the serum of sheep in the group of vaccine with 1%levamisole adjuvant reached up 1:128 and 1:512 separately after the first and second immunization.Conclusions could be drawn that both two inactivated vaccines could promote proper immune effects and inactivated vaccine with aluminum hydroxide adjuvant had better immune effects.