Studies On The Virus Propagation And The Quality Criterion Of Main Materialsused In Inactivated Avian Influenza Vaccine

It has been attracted high attention to the high pathogenic avian influenza (AI) broken out and spread in the world. The effective prevention and control methods for the disease generally include the quarantine blockade, killing the infected and threatened animals and other biological safety measures. At present, some countries and regions have eradicated m the epidemic high pathogenic AI by using those methods. However, when the epidemic disease spread wildly in some areas, it could not be prevented and controlled effectively by using the above measures, and it could cause great economic loss. And the vaccination management must become the one of the key measures to control the epidemic disease. In China, vaccination has been used as the one main measure of the comprehensive prevention system for avian influenza. The vaccine was produced by8bio-products factories and the marking of the products was arranged by Chinese government under the invited public bidding and stock system. The preparation, sale, administration of the vaccines and the development of the new vaccines against high pathogenic AI were administrated and inspected strictly by Chinese government. It has caused great attention to the quality of the vaccines from the state, the manufacturing enterprises and the animal breeders.In order to improve the problems in safety, protective efficacy, stability and homogeneity of the current vaccine against AI, it was firstly studied on the quality criterion of seed eggs, paraffin injected and viscosity in this study by followed the two items administrated by the4th Veterinary Medicine Pharmacopoeia Committee, the quality criterion and inspection criterion for the injected paraffin and the criterion and the method for a substitute viscometer for straw. And the criterion of the vaccine production in the biological industry and state was formulated. It should be helpful for increasing the quality of the AI vaccine and satisfying the need of the internal and international markets. The content of the study are composed of three parts as following: 1. Studying on the method for propagation of avian influenza virus and the quality control of the seed eggs for productionBased on the levers of the maternal antibodies against AI of the seed egg from the chickens unvaccinated with AI vaccine, the optical concentration of the virus and the optical egg-embryo age for inoculation were titrated by the matrix method. The result showed that:(1) when the titer of the maternal antibody against AI was less than2Iog2, the optical diluted concentration of virus was1:1000(10EID50), the optical egg-embryo age for inoculation was10days old, the quantity of the harvested allanoic liquid for each egg was up to12.5ml, the average HA titer was111og2, and the virus titre was108.5EID50/0.1ml;(2)when the titer of the maternal antibody against AI was less than6log2, the optical diluted concentration of the virus was1:10000(104EID50), the optical egg-embryo age for inoculation was10days old, the volume of the harvested allanoic liquid for each egg is up to12ml, the average HA titer was121og2, and the virus content was108.8EID50/0.1ml. To make sure that the accuracy of the experiment, all the inoculation and harvest of the egg-embryos allanoic liquid were conducted by the automatic inoculation and harvest machine system. Analyzing the above data comprehensively, the best method for the propagation of the virus was selected as:the10days old egg embryos was inoculate by using virus dilution of1:10000(104EID50).According to the above optical condition for virus propagation,14batches of the inactivated vaccine was produced in total5.445billion milliliter, by using2300thousand eggs provided by14main seed egg supplier for making AI inactivated vaccine. By the statistic analysis of the appearance,weight, dilapidation rate, the fertilized rate, dead embryo rate, utilization ratio and harvesting quantity14seed eggs unvaccinated, the internal quality control criterion of the seed eggs for production was established. The14batches of seed eggs appeared clean, homogenized in size chosen by chosen machine, weight of50-60grams, the dilapidation rate being lower than2%for10of14batches, the fertilized rate of all the eggs being over91%, dead embryo rate before inoculation being lower than4%for11of14, utilization ratio being over85%for8of14batches. Making a comprehensive evaluation of the dead embryo rate post inoculation, harvesting quantity per embryo, and the virus titer index, it showed that the dead embryo rate was below10%, harvesting quantity per embryo was above10ml, the virus titer was over101og2. Among the14batches of vaccine against avian influenza, llbatches had the HA titer of81og2, which being higher than the criterion of61og2. It indicated that the various indexes of the seed eggs were reasonable, which being available to guarantee the quality of the vaccine.2. The study of the quality criterion and the Standard Operation Procedure of the paraffin injectedOne type of imported paraffin oil MC, used popularly in the8fixed manufacturing enterprises of vaccine against AI, and two types of paraffin, LH and HZ, made in China with different viscosity are chosen and the quality was detected as the method for the quality criterion of the paraffin injected and the paraffin oil for food in China Veterinary Pharmacopoeia (2005edition), GB48532008. The results showed that the3different sources of paraffin had different movable viscosity, the content of condensed aromatics, and the distribution of the carbon numbers. The4LH with different origination had the lowest movable viscosity and the content of condensed aromatics, the least narrow of distribution of the carbon numbers and the distinct of each component, which being4.37-5.7mm2/s、0.005-0.015and15-19, respectively. The3indexes of the paraffin in MC were7.26mm2/s、0.162and15-24, respectively. The3indexes of the paraffin in HZ were11.16mm2/s、0.037and15-24, respectively. The result of the safety experiment demonstrated that the absorbing of the vaccines with different origin paraffin were different. When inoculation via muscle route, the vaccine with LH which originated with5.7mm2/s of movable viscosity could be best absorbed completely48days post inoculation. Meanwhile, the vaccine with HZ was less absorbed. And one bird had a few unabsorbed dry cheeses like materials in the injection area. In the group MC with over the standard of the content of the condensed aromatics, the vaccine with HZ was also less absorbed and there were severe local inflammation. The result of the efficacy test indicated that the levels of HI antibodies to AI in LH groups were higher than those in the MC and HZ groups, which being7.61og2and9.51og2at2and3weeks post inoculation. The HI antibody titer was dramatically higher from ZSLHAI vaccine than that from ZSMC、ZSHZ vaccines, which remaining9.41og2at130days post immunization. After the vaccines was stored for12months and inoculated with chicken, the HI antibody titer from ZSLH vaccine was8.5log2. But when ZSMC、 ZSHZ vaccines were preserved for9months, they only could provide61og2HI antibody to AI.The results of immunological protection efficiency for different strains of chickens and ducks indicated as following:(1) when the commercial7days old broilers were vaccinated with ZSLH, ZSMC, ZSHZ, respectively, the antibodies were detected at28days post inoculation, which being7.81og2for the ZSLH group,6.71og2for the ZSMC group, and6.11og2for the6.11og2group.(2) When the18days old egg-laying chickens were vaccinated, the antibody reached the peak at4weeks post inoculation (wpi), which values for ZSLH, ZSMC, ZSHZ being7.11og2,5.41og2and5.21og2, respectively.(3)When the18weeks old breed layers were vaccinated, the antibody reached the peak at1month post inoculation, which values for ZSLH, ZSMC, ZSHZ being10.81og2,9.91og2and8.21og2, respectively.(4)When the commercial10days old meat-type ducks were firstly vaccination, the antibody reached the peak at4wpi, which values for ZSLH, ZSMC, ZSHZ being7.11og2,5.41og2and5.21og2, respectively. As the second enhancement at3weeks old, the antibody reached the peak5wpi, the values being9.31og2,8.31og2and7.91og2for ZSLH, ZSMC, ZSHZ, respectively.(5)When the susceptible14days old egg-laying ducks were vaccinated, the antibody reached the peak at5wpi, the value being8.51og2for the ZSLH group, but in ZSMC and ZSHZ groups, the antibody reached the peak at4wpi, which value being5.31og2and5.11og2, respectively. After the second enhanced vaccination at3weeks old, the antibody reached the peak at6wpi, which values being10.81og2,9.41og2and9.11og2for ZSLH, ZSMC, ZSHZ, respectively. It indicated that the low viscosity, low content of condensed aromatics and the purity of paraffin LH made in China were superior to those of the imported and the similar product made in China. The vaccine made with paraffin LH had higher safety and immunological protective efficiency than those with other oil adjuvant.3. Studies on the method of a substitute viscometer for straw for detecting the viscosity of inactivated avian influenza vaccineThe viscosity of the adjuvant was the key factor affecting the viscosity of the vaccine. In this study, the viscosity of the vaccines made with3different paraffins was detected by using a method of a substitute viscometer for straw. The results showed that when no change on the manufacturing procedure and the prescription, the viscosity of the vaccine made with paraffin LH, which viscosity being4.3-5.0mm2/s, was the lowest (≤40mPa.s). Meanwhile, the viscosity of the vaccine made with paraffin MC, which viscosity being7.31-7.42mm2/s mm2/s, was less109mPa.s. And the viscosity of the vaccine made with paraffin HZ, which viscosity being8.48-11.29mm2/s, was lower than166mPa.s. It indicated that viscosity of the vaccine could be decreased dramatically by changing the viscosity of the oil adjuvant.The prescription was also the important factor for the viscosity of the inactivated avian influenza vaccine. By using the same oil adjuvant, when the volume rate of the water phase to the oil phase was1:3, the viscosity of the vaccine was not higher than60mPa.s by using aluminium stearate, but not higher than40mPa.s without aluminium stearate. When the volume rate of the water phase to the oil phase was1:1.5, the viscosity of the vaccine was the highest,165mPa.s, by using aluminium stearate, but119mPa.s without aluminium stearate. After detection of8commercial vaccines, the results showed that the viscosity of the vaccines produced by different manufacturers were different. The lowest of the viscosity was18.7mPa.s and the highest was91mPa.s. Moreover, among the8commercial vaccines,6types of vaccines had low viscosity of less60mPa.s. It indicated that no more than60mPa.s could be used as the internal viscosity criterion for the inactivated avian influenza vaccine in industry. Finally, after the viscosity of64oil-emulsified vaccines samples were detected with the method of viscometer and the straw, the regression model was established. And it was found that there were significant regression relation between the method of viscometer and the straw.In summary, because the quality of the raw materials was the key factor affecting the quality of the avian influenza vaccines, it was necessary to establish the internal control quality criterion of the seed eggs and the quality criterion of the paraffin injected for making sure the vaccine safe, stable, highly efficient and homogeneous. Secondly, the low viscosity, content of the condensed aromatics and high purity of the paraffin were helpful for producing the influenza vaccines with high safety and immune efficiency, antigen stability and the period of immunity. Third, the method viscometer could substitute for the method straw for detecting accurately the viscosity of the vaccine, which being in line with the international standards. The viscosity of the adjuvant and the prescription were the important factors in affecting the viscosity of the inactivated avian influenza vaccine.