Isolation,Identification And Preliminary Identification Of The Bacterial Ghost Vaccine Of Bovine Capsular Serotype A Pasteurella Multocida

Objective: The etiology of bovine respiratory disease is complicated which caused huge economic losses for cattle industry.The disease was initially related to transportation factors, also known as "shipping fever",but transportation factors is just an inducement,bacteria and viruses are the main causes of the disease, and there is a complex interaction between bacteria and viruses,Pasteurella multocida is one of the important bacterial pathogens. Bovine respiratory disease has emerged and caused some economic losses in some areas of Xinjiang since 2006, We isolated and identified bacteria with conventional bacterial isolation method combined molecular biology separation method based on bacterial 16 Sr RNA. Phage gene E was Cloned, constructed lysis plasmid which was induced expression and bacterial ghost was preparedMethod:1 、 We isolated bacteria from tissue lesions conventional bacterial isolation method combined molecular biology separation method based on bacterial 16 Sr RNA. Bacteria serotype was identified with PCR based on gene Kmt-1 and serotype-specific genes（hya D-hya C、bcb D、dcb F、ecb J、fcb D）. Drug sensitivity and LD50 of the isolates were tested by K-B disk diffusion method and convention mathod.2 、 The lysis gene E of phage was cloned into T vector, and prokaryotic expression vector p BV220-E, p ET28a-E were constructed which were transformed into Pasteurella multocida,then induced expression.bacteria,bacterial ghosts was observed with a scanning electron microscope and preliminary tests were carried out in mice.Results:1 、 Strain of serotype A Pasteurella multocida were isolated and identified.16 S r RNA sequence of Pm have the homology of specific 16 S r RNA gene fragment were over 99 %compared with AY299308（Scotland）, JQ404476（Xin Jiang）, NR₀41809（USA） from genbank.Isolate was resistant to clindamycin, Intermediary to streptomycin, gentamicin, kanamycin and susceptible to tetracyclines, quinolones, cephalosporins, penicillin.All the infected murines died within 5.5h,LD50 was 1.48 CFU/m L.2、Vector p BV220-E,p ET28a-E were constructed expression successfully,but expression vector p BV220-E can not transforme into Pm. Expression vector p ET28a-E was successfully transformed into Pm, which induced expression to the bacteria ghosts; Protective rate to mice of bacterial ghost was 40%, protection rate add Freund’s adjuvant was 60%.Conclusion: Serotype A Pasteurella multocida was isolated and identified which was the one of main pathogenic bacteria from the farm. Bacterial ghost was prepared and it’s had certain protection rate in mice.