Preparation Of IBR-BVD Double Inactivated Vaccine And Study On Its Immune Effect

Infectious bovine rhinotracheitis（IBR）and bovine viral diarrhoea/mucosal disease（BVD/MD）are two common viral infectious diseases prevalent in herds.BVD and IBR often appeared clinically at the same time.The vaccination is the primary method to prevent and control these two infectious diseases,however,at present there is still no commercialized vaccine in the domestic industry.So it is badly in need to develop a safe and effective vaccine for preventing BVD and IBR simultaneously.In order to screen for the candidate strains of IBR-BVD combined killed vaccine,it is necessary to research on the physiochemical properties,including the acid sensitivity,of the IBRV DQ-1 strain and BVDV HJ-1 respectively,trypsin sensitivity,heat resistance,diethyl ether sensitivity,Mg²⁺protectiveness,chloroform sensitivity and so on.To obtain the multiplication techniques of the two viruses,we explored the inoculation method,tampering with medium selection,serum content,exposure dose,time of harvest,number of the freezing and thawing procedures,etc.To prepare the combined inactivate vaccine,we inactivated the two viruses by formaldehyde,optimized the formaldehyde concentration,inactivation time as well as the inactivation temperature,selected the Montanide ISA 206 as adjuvant and white oil adjuvant,emulsified 8 single vaccines and combined inactivate vaccine in accordance with the different virus tilter and adjuvant and then tested the prepared vaccine according to the requirements of the Chinese Veterinary Pharmacopoeia.We divided 54 pregnant dairy cattle ranging from 18～30 months of age randomly into 14 groups for immunity test,the second immunization took place 21 days after the first one,observed the clinical symptoms.Before the immunization,7,14,and 21 days after the first immunization,and 7,39,and 99 days after the second immunization,we got blood from the caudal vein of the tested herds and separated the serum.Finally,we evaluated the immune effect with the virus neutralization test.The result showed that the two viruses were both sensitive to diethyl ether,chloroform and pancreatic enzymes,and had weak resistance to acid and heat,Mg²⁺doesn’t have protective power for them.The best cultural medium for both of them is DMEM.Respectively,the optimum serum content in growth promoting medium and maintenance media are 8%and 0%respectively,the best times of freeze and thawing is 2,the best inoculation dose for IBRV and BVDV are 0.02 MOI and 0.04MOI,the best inoculation ways for IBRV and BVDV are synchronization and sensation,and the best harvest time for IBRV and BVDV are 80%CPE and70%CPE.The best inactivation condition for IBRV is to act 12h in 37℃air table（100 r/min）with 2%of formaldehyde concentration,while the best inactivation condition for BVDV is to incubate 4h in 37℃air table（100r/min）with 0.25%of formaldehyde concentration.The prepared 8 single vaccines and combined inactivate vaccine both confirm with the relevant requirements in Chinese Veterinary Pharmacopoeia.We tested the inactivate vaccine in each group of dairy cattle.After the first immunization nothing abnormal detected in each tested group.After the second immunization,the temperature of the group with IBRV(10^7.5TCID₅₀/0.1mL)-BVDV(10^6.2TCID₅₀/0.1mL)white oil adjuvant combined inactivate vaccine rise to around 41℃,4 days after the second immunization the temperature resume to normal level while the other groups keep normal.Apart from the BVD(10^5.2TCID₅₀/0.1mL)group,which yielded no antibodies,the other groups of vaccine all induced the dairy cattle to produce neutralizing antibody,the peak value in those groups appeared 39 days after the second immunization with 1:98.5 for IBRV and 1:78.1 and BVDV.Neutralizing antibodies were still detectable 99 days after the second immunization.The IBRV and BVDV antibody level rise up along with the increase of immunizing doze.The group with high dose（4mL/cattle）has a better security for the tested animals as no pregnant dairy cattle had an abortion.A IBR(10^7.5TCID₅₀/0.1mL)-BVD(10^6.2TCID₅₀/0.1mL)206 immunization group was selected.The production of single vaccine and combined inactivate vaccine that have good security and can produce a certain degree of neutralizing antibody valence has laid a solid foundation for the next step of virus attack test.