Epidemiological Investigation Of Mycoplasma Bovis And Development Of Inactivated Vacciue

Mycoplasma bovis has not been paid enough attentionsince its discovery.It is not listed as major diseases,but lossescaused by Mycoplasma bovis in cattle breeding are always great.Reports have suggested that Mycoplasma bovis-causeddiseasesareglobally distributed,and bovine mycoplasma has strong pathogenicity causing bovine mastitis and pneumonia.In this study,Mycoplasma bovis strains in most regions of China were isolated and identifiedand its epidemiological investigation was conducted.Based on the depuration and serial subcultivation,inactivated vaccineof Mycoplasma bovis and indirect Enzyme-Linked Immunosorbent Assay(ELISA)detection method were studied.During the past eight years,epidemiological investigation of Mycoplasma bovis was conductedon 30 large-scale dairy farms in 12 provinces of China using Cultivation,indirect ELISA,and Polymerase Chain Reaction(PCR)detection methods.The results showed that showed that the positive rate of Mycoplasma bovis antibodyin blood serumwas 52.31%(441/843);the positive rate of tissue and swab samples was 55%(33/60);the positive rate of milk samples was 34.22%(373/1090);the 16s rDNA sequences of 55 representative Mycoplasma bovis strainsshowed that no mutation occurred at the 16srDNA level.55 Mycoplasma bovis strains of mycoplasma were used for the research of biochemical characteristics,growth inhibition,nutrition and metabolism and drug sensitivity test,and PCR,color change unit(CCU),colony forming unit(CFU)were used to obtain data.Then dominant strains were serial subcultured.For now,viable count of Mycoplasma bovis has stably reached to 1011 CFU/mL,and 200 in vitro continuous passage cultureshave been conducted.Ultrasonic lysate of Mycoplasma bovis tropinawas used as coating antigento establish indirect ELISA detectionmethod.Results of orthogonal screening showed that the proper protein coating amount is 4 μg/mL,the proper coating liquid is carbonate buffer(pH 9.6),the optimum coating temperature and time is 4℃for 16h,confining solution is 1%skimmed milk,the lutationtemper atureandtimeis 37℃ for 2h,the optimal serum dilution is 1:100,the reaction temperature and time is 37℃ for 1 h,the secondary antibodies dilution is 1:4000,the optimum reaction time and temperature is 37℃ for 1h.The critical value of Mycoplasma bovis antibody-negative serum is 0.346(OD450).The indirect ELISA detecting method showed great specificity and no cross reactionsoccurred withthe positive serums of foot-and-mouth disease,paratuberculosis,brucellosis,bovine viral diarrhea,infectious bovine rhinotra cheitis and tuberculosis.When the positive serum was diluted to 1:512,the OD450 valuewas still higher than the critical value,which proved that the method has good sensitivity.The coefficient of variationamong groups and within groups were less than 5%,which proved that the method has good reproducibility.150 clinical samples were detected using our indirect ELISA detecting method and indirect ELISA kit for Mycoplasma bovis(Canadian Biovet),the results showed that the coincidence rate of two methods is 95%.These results suggest that the indirect ELISA method established in this study can be applied asa method for rapid diagnosis,epidemiological investigation and detection of antibodies of Mycoplasma bovis.Purified Mycoplasma bovis strains were used for the identification of virulence in rabbits,the symptoms were highly similar with that of Mycoplasma bovis infection in cattle.The results of pathological tissue sections,indirect immunofluorescence,PCR and isolation methods demonstrated that they were Mycoplasma bovis infection.The results also showed that the virulence of Mycoplasma bovis was obviously weakened,but it was still pathogenic.Thus,inactivated 160th generation of Mycoplasma bovis was mixed with aluminum salt adjuvants with a ratio of 1:1 to immunerabbits,and the rabbits were infected at the 28th day.Rabbits in the vaccine group showed no obvious pathological changes,rabbits in the control group had fever,lung lesions were obvious after dissection.The rabbit immunity test showed that the inactivated Mycoplasma bovis vaccine could produce a proper protective effect.Based on rabbit immunity test,inactivated vaccine of Mycoplasma bovis and immune vaccination program were clinically appliedfor immunizing 210 bullocks(60 days)on a cattle farm in Inner Mongolia.Live bacteria content in inactivated vaccine was 1011CFU/mL,and the immunized dose was 1mL.At the 60th day,as for bullocks in the vaccine group,the incidence rate decreased by 20.7%,strain isolation rate decreased by 35%,the positive rate of PCR decreased by 50%;as to the bullocks in the control group,the incidence rate increased by 17%,strain isolation rate increased by 8.3%,the positive rate of PCR decreased by 25%.The antibody titer after two vaccinationsreached up to 1:256,antibody titer of 80%of bullocks reached to 1:128.The antibodies were still detectable and the antibody titer of 67%of bullocks reached up to 1:64 at the 60th day.The optimal immune time,immunization program and the antibody fluctuation regularity were determined.