Isolation And Identification Of Bovine Respiratory Mycoplasma And Establishment Of Rapid Diagnosis Method Of Important Pathogens

In recent years,bovine respiratory diseases have become one of the main factors that harm the cattle breeding industry in China.Especially in winter and long-distance transportation,incidence rate and mortality rate increase greatly.Bovine respiratory syndrome is mainly caused by Mycoplasma bovis,pasteurella and bovine infectious rhinotracheitis virus.If it is not diagnosed in time,it will delay the disease and even cause the death of cattle.Therefore,the development of rapid diagnostic methods can provide important technical means for the accurate prevention and treatment of bovine respiratory syndrome.The main contents of this study are divided into three parts:1.Isolation,identification and drug sensitivity test of Mycoplasma bovis.2.The detection method of Mycoplasma bovis fluorescent RAA was established and optimized.3.Based on the OPPF gene of Mycoplasma bovis,kmt-1 gene of Pasteurella multocida and g B gene of bovine infectious rhinotracheitis virus,a multiplex PCR method was established,and the conditions were optimized and evaluated.The results showed that five strains of bovine mycoplasma,named zyt-1-zyt-5,were isolated and identified from the lungs of dead cattle by staining microscopy,culture characteristics,specific gene detection and 16S r RNA sequence comparison.The drug sensitivity test showed that the isolates were extremely sensitive to enrofloxacin,ciprofloxacin,oxytetracycline and florfenicol,and extremely sensitive to hematoxylin,Schisandra chinensis,Pulsatilla chinensis and dark plum;The established fluorescent RAA detection method for Mycoplasma bovis can be completed at 35?and 20 min when the probe concentration is 1.6?mol/L,primer concentration 0.8?mol/L,the minimum detection limit of Mycoplasma bovis template was 4.2 pg/?L;The specificity of RAA detection method was 100%,the coincidence rate with conventional PCR method was 100%,and it had good repeatability.The established multiplex PCR method for Mycoplasma bovis,Pasteurella multocida and infectious bovine rhinotracheitis virus had the optimal annealing temperature of 55?,the optimal number of cycles of 30 and the optimal primer addition of 0.5?mol/L,the optimal template concentrations of Mycoplasma bovis,pasteurella and infectious bovine rhinotracheitis virus were respectively 3.9×10^-3ng/?L,6.61×10^-2ng/?L,2.97×10^-2ng/?L.the specificity was 100%,and the coincidence rate was 100%compared with single PCR.In conclusion,five strains of Mycoplasma bovis were isolated and identified in this study.Antibiotics sensitive to Mycoplasma bovis and single Chinese herbal medicine were screened.The fluorescent RAA detection method of Mycoplasma bovis and the triple PCR detection method of Mycoplasma bovis,Pasteurella multocida and infectious rhinotracheitis virus were established for the first time.The established RAA detection method and triple PCR detection method of Mycoplasma bovis can be used for the detection of mixed infection and single infection of Mycoplasma bovis,pasteurella and infectious bovine rhinotracheitis respectively,so as to provide scientific and technical support for the prevention and treatment of bovine respiratory diseases.