Establish Of Purification And Concentration Technology For Newcastle Disease Vaccine(La Sota Strain)

Newcastle disease(ND), caused by Newcastle disease virus(NDV), is an important acute septic infectious diseases in poultries, which bring huge economic losses for poultry industry.Vaccination can effectively prevent and control the incidence and prevalence of ND and reduce economic losses. At present, there are two kinds of commercial vaccines in markets, attenuated vaccine and inactivated vaccine, respectively. The live vaccine includes Class I, Class II(B1 strain), Class III(F), Class IV(La Sota strain) and Clone 30. Among them, Class IV(La Sota strain) is the most commonly used attenuated vaccine. Moreover,long-term application results indicated that La Sota strain had good immune effect and could protect chickens against NDV challenge. In the process of ND attenuated vaccine production, the traditional method includes the inoculation of SPF chicken embryos, the collection of allantoic fluid, the freeze-dried process after mixing with a certain percentage.The vaccine not only could cause some adverse reactions, such as swollen head,indigestion, inappetence, local inflammation, shock and even death, but also resulted in lower performance, immune function reduction, easy onset, response reduction to other vaccines. The adverse reactions produced by the vaccination could be caused by a variety of factors, including endotoxin, exotoxin, heat, variant protein, egg source, serum,gram-negative bacteria and the environment. Among them, the endotoxin is one of the main factors. Therefore, in the ND vaccine(La Sota strain) production process, the optimization of intermediate production processes could reduce and avoid the endotoxin contamination in the vaccine, and further enhance the quality of the vaccine, and improve the safety and effectiveness of the vaccine.Experimental design was four groups consisting of group A, group B, group C and group D.For group A, group B and group C,the vaccine was produced in concentration and purification manufacturing technique with continuous production of three batches of production, For group D the vaccine was produced in original antigen as the control group.The virus was diluted by a certain appropriate dilution and then was inoculated to SPFchick embryo allantoic cavity with 0.1 m L / embryo. The virus that had been inoculated was set in 36 to 37 ℃ to hatch. The chick embryoes that died within 72 hours were discarded when harvest.The allantoic fluid of chick embryo that survived in 96 h was collected and was detected the hemagglutination titer and EID50,.Some of the qualified samples was concentrated with Pel ultrafiltration system and was produced according to the inspection procedure marked as experiment group A, B and C.respectively. Another part of the antigen were produced according to the inspection procedure of vaccine marked as control group D. The four groups of products were produced for continuous three patches and detected following test: endotoxin test, sterility test, determination of virus, safety inspection and immune and protection test. The concentration and purification technique of chicken Newcastle disease live vaccine was then established by comparing the above result.As the results showed that the 10/10 safety detection of Newcastle disease live vaccine(La Sota strain) of the experimental group A、B and C were qualified, the endotoxin concentration was not exceed 20EU/ m L,the detection quantiation of virus in this vaccine was greater than 106.0 EID50; As the compared, the 7/10 safety detection of Newcastle disease live vaccine(La Sota strain) of the experimental group D were qualified, the endotoxin concentration was exceed 50EU/ m L and the quantiation of virus was greater than 106.0 EID50.By comparing the detection results of experimental group with the control,we can get a conclusion that the production process of concentration and purification was necessary.Controling the qualify of raw and auxiliary materials and the production process can not only validly accmulate and regulate the quantiation of virus solution,enhance the active ingredients of vaccine,reduce the volume of used vaccine,but also can remove some ingredients of bio-derived impurities,reduce the endotoxin contamination of virus solution in semifinished product and the difference between batches,the adverse effect of vaccine inoculation.So that can promote the level of the product qualify.This experiment had estabished the production process of concentration and purification of Newcastle disease live vaccine(La Sota strain),It is expected that can be applied in the future production.