Studies On An Inactivated Vaccine Against Bovine Colibacillosis And Pasteurella Multocida

Diarrheagenic Escherichia coli?E.coli?is a main pathogen of neonatal calf diarrhea?NCD?,which has a great influence on the development,growth and survival of calves.Bovine Pasteurella multocida?PM?mainly caused bovine systemic hemorrhagic septicemia,bovine respiratory systemic and calf diarrhea by mixed or secondary infection,and acute onset occasionally.Mixed infection were often occured between these two kinds of bacteria,and exacerbating the occurrence of diarrhea.The two bacteria were widespread in cattle in our country,caused huge economic lo sses to the dairy and beef industries.Currently,there have no commercialized combined vaccine against Bovine colibacillosis and Pasteurella multocida.In this study,we use the bovine E.coli and bovine PM,which isolated from calf with diarrhea,to develop a combined vaccine against Bovine colibacillosis and Pasteurella multocida.For this purpose,we identified the bacteria and established the Seed Lot of bacteria.Moreover,we explored the conditions of production process,improved the quality characteristics of vaccines,providing data for the industrialization of vaccines.First of all,E.coli?O101:K99?and PM?capsular B?,named E0803 strain and P0604strain respectively,were isolated from calves with diarrheal diseases.?The virulence test showed that the LD₅₀ of strain E0803 was 9×10⁶CFU in mice,but had no virulence to New Zealand rabbits.The minimum lethal dose?MLD?of strain P0604 to mice and New Zealand rabbits was lower than 10 CFU.Immunity experiment showed that the protection rate of strain E0803 could get 80%?8/10?,while strain P0604 could get100%?10/10?,compared with the control group?10/10 death?,suggesting that all two strains had good immunogenicity and could be used as vaccine strains.Seed Lot of the two strains were estabished,and the biological characteristics between different generations of strains were determined.The results showed that the culture characteristics,morphology and purity of the two strains?F1^F20?were consistent with each other.The biochemical characteristics,virulence and immunogenicity of the two strains?F1\F10\F20?were not significantly different.In practical application,it was suggested that F1^F4 of the two strains should be taken as the original seed generation,F5^F10 was used as the basic seed generation,and the seed subculture was controlled within 5th.Selected the culture medium,time of culture,amount of serum added during culture,conditions of large production process,inactivation process and emulsification process.It was determined that the best medium for both strains was TSB.In the process of large scale fermentation,the culture conditions of both strains were as follows:10 h at 37?,0.03 MPa under pot pressure.Among them,stirring speed can be adjusted 200 r/min for strain E0803,100 r/min for strain P0604.At the same time,2%⁵%of serum was added to culture the PM.0.2%of formaldehyde solution was added into bacterial broth,inactivated at 37?for 24³6 h,was the best inactivation condition.The ratio of water and oil was 1:2,the emulsifying condition of small emulsifying machine was 8000r/min for 5 min,and 4000 r/min for 20 min of large emulsifying machine.New Zealand rabbits and mice were used to security check of the combined vaccine.The results showed that the combined vaccine was safe for both rabbits and mice after overdose?2 times?once and single dose repeated inoculation.There was no adverse reaction caused by vaccine in experimental animals after inoculation.In order to establish a method for detecting the efficacy of the bivalent inactivated vaccine,mice were immunized and then challenged,instead of the target animals.The results showed that the mice could develop immunity on 14^th day after immunization.The immunized mice could get more than 80%protection after challenged on the 21th day,which indicated that the combined vaccine against Bovine colibacillosis and Pasteurella multocida was safe and effective.Our research provided scientific experimental data for the development of inactivated combined vaccine against Bovine colibacillosis and Pasteurella multocida,and provided the basis for the industrialization of the vaccine.