Observation On The Growth Characteristics Of Mycoplasma Bovis And Animal Immunization Test

Mycoplasma bovis is an important pathogen causing pneumonia,mastitis,otitis media,genital tract inflammation,arthritis,and corneal conjunctivitis in cattle,which have been designated as Mycoplasma bovis-associated disease?MbAD?.M.bovis is popular all over the world,which has caused huge economic losses to the cattle industry.The biological characteristics of 19 strains of M.bovis were observed,and the growth characteristics and immunogenicity of strain 08M were further studied.19 strains of M.bovis were inoculated in the Thiaucourt's medium for about 12 h,the color of the medium changed from red to orange and the pH value decreased to about 6.8 from 7.4.After inoculated in the solid medium for about 3 days,a typical"fried-egg"colony for every strain was observed under the microscope.The total proteins of all M.bovis strains were extracted and analyzed by SDS-PAGE and Western-blot.The results showed that the size and number of the total proteins of these strains were almost same,and the numbers of protein bands were 18²0.The Western-blot incubating with 08M strain immune serum results showed that the size,numbers and intensity of the immunized bands of 19strains of M.bovis were consistent.Multilocus sequence typing?MLST?was used to analyze 15Mycoplasma bovis isolated from China,finding that 15 Mycoplasma bovisall belonged to ST10.Based on these results and the previous pathogenicity test of strain 08M in the laboratory,the growth characteristics and immunogenicity of the strain 08M were further studied.M.bovis strain 08M was cultured in the Thiaucourt's medium?The volume ratio is 3:5?at an initial inoculation amount of 70 CCU/mL.Four growth curve plotting were made by measuring color change units?CCU?,colony forming units?CFU?,protein concentration and nucleic acid levels.Both the results of CCU and CFU counts showed that the growth of strain 08M was divided into four phases:the logarithmic phase appeared after being cultured for 10 h;the stationary phase appeared at 30 h with the highest number of viable cells up to 10⁸ CCU/mL and 7.7�10⁷ CFU/mL,and the decline phase started from 75 h.The protein concentration of M.bovis increased rapidly from 15 h to 35 h,reached 72.06?g/mL at 35 h,then kept between 58.38?g/mL and 70.65?g/mL.The nucleic acid level of M.bovis increased rapidly from 15 h,and the cycle threshold?Ct?values were maintained between 15.32 and17.84 after 25 h.The cells of strain 08M were collected at the end of the logarithmic growth phase.After washing three times with p H 7.2 PBS,the cells were diluted into two different concentrations,and inactivated with BEI,and then emulsified with MONTANIDETM ISA 201 VG to prepare two inactivated vaccines which antigen concentrations was 0.74 mg/mL or 0.23 mg/mL,respectively.The 8⁹ months old calves was subcutaneously injected with 2 mL of the inactivated vaccine per calf,and boosted at 28 days after the first immunization.The negative control group and the blank control group were also set.The challenge test was conducted at 28 days after boosted.The results showed that both two inactivated vaccine could produce high levels of antibodies after immunization,All tested cattle were dissected at 4weeks after challenge.different degrees of lung lesions were observed and M.bovis were isolated or PCR positive from all animals.It indicated that vaccination with 2 preparations with different concentrations of strain 08M could result in a strong antibody response but did not protect calves from an experimental challenge.