Development Of Real-time PCR For Detection Of Infectious Bovine Rhinotracheitis And Development Of Inactivated Vaccine

Infectious bovine rhinotracheitis(IBR) is an acute,heat and contagious disease caused byinfectious bovine rhinotracheitis virus(IBRV).Its main clinical manifestations aredyspnea,rhinitis and dams abortion.Although the mortality rate caused by the disease is low,thereis a serious impact on fattening, milk production and international trade.It is necessary toestablish a sensitive,rapid and reliable diagnostic method and develop inactivated vaccineforpurify the herd.Due to the absence of a suitable animal model for the evaluation of immunieefficacy,the evaluation of conventional vaccine is still difficulty.This study is preliminary studyof the infection of health rabbits and vaccined rabbits with IBRV.It is pregnant for the evaluationof vaccine which is used for the control of IBRV.The study developed the TaqMan-MGB probe real-time PCR for detection of infectiousbovine rhinotracheitis.A pair of specific primers and the MGB probe were desgined based onsequence of IBRV Bartha Nu/67in GenBank. Thecorrelation coefficient of the standard cure was0.998.The detected limit of the method was1.49×101copies/μL.After clinical examination,themethod is suitable for rapid quantitative detection of clinically suspected samples.The17nasal swabs of suspected cow were collected,and inoculated to MDBK.They wereidentified by PCR,immunofluorescence test,micro serumneutralizationexperimentand genesequence homology analysis.One strain was determined IBRV and named IBRV DQ/14strain.Then,the rabbits were infected with IBRV DQ/14of10-7.33/100μL TCID50.The nasal swabs of1-14d were collected and serums were also collected every7day.There were the autopsy andcollection of tissue organs after inoculation3d,5d,7d,11d and49d.The results showed that therabbits occoured clinical symptoms and detoxification last7d,a large number of IgG wereproduced during7-14d.Histopathological and immunohistochemical showed that inflammationoccurred in lungs and the virus replicatied in epithelial of turbinate and lung.The studies haveshown that IBRV DQ/14is pathogenic for rabbits.IBRV DQ/14was inactivated by BEI and the Montanide ISA206adjuvant was used toprepare a strain inactivated vaccine.The Balb/c mice and pups were injected vaccines for thesafety testing. Rabbits were immunitiedand the same dose of a strengthening immunization after 21d.Another14days later,the rabbits injected with IBRV DQ/14.The result of showed that thevaccine prevented the colonization of virus in the respiratory tract colonization, the protectionrate is100%.In summary,thisstudy islated a strain IBRV,developed the TaqMan-MGB probe real-timePCR for detection of infectious bovine rhinotracheitis,proved IBRV DQ/14is pathogenic forrabbits.It provided a quick detection method and an inactivated vaccinefor the prevention andcontrol of IBRV.