| Infectious bronchitis is a common acute disease caused by infectious bronchitis virus (IBV). The virus has an envelope structure, and is particularly sensitive to high temperature, which places additional difficulty to the development of a heat resistant protective agent for infectious bronchitis vaccine production. Due to the viability of the virus easily influenced by high temperature, after freeze-dried, vaccine strain titer loss will become evident, which forms a bottleneck for improving of freeze-dried infectious bronchitis vaccine product, this is also a universal problem faced by the poultry vaccine industry. Domestic conventional freeze-dried vaccine must be kept below-15 ℃, and the storage period is short, while foreign freeze-dried vaccine products can be stored at 2-8℃ for more than two years, and at a high temperature over 2-8℃ can also be kept for a long time. In order to reduce the freezing-dried vaccine strain loss, improve the quality of the vaccine, prolong the shelf life and solve the problem of cold chain transportation, the IBV H120 strain heat resistant protective agent developed in this study can provide some help.The content of this study is divided into three parts, namely, the screening of heat resistant protective agent and the optimizing of the curve parameters during freeze drying; the anti-aging test of heat resistant protective agent after screening; the efficacy study of heat resistant protective agent freeze-dried vaccine.In the heat resistant protective agent component screening experiments, a total of 8 different formulae were tested, numbered from A to H, where the C formula was divided into C1-C6, D formula was divided into D1, D2, respectively. After being freeze-dried in eight groups, products were compared for frozen form, solubility, residual moisture content and virus titre loss. In the end, we screened the best E formula as heat-resisting protective agent, the main components are poly-peptone and trehalose. An optimized freeze drying curve is also essential to improve the quality of the freezing-dried vaccine, that each formula of heat resistant agents must be corresponding to a suitable freeze drying curve. First, a small amount of vaccine was freeze-dried with different heat-resistant protectants. According to the morphology of the lyophilized product, the freeze-drying curve parameters were optimized, such as the pre-freezing minimum temperature, sublimation drying temperature and vacuum degree and so on. Ultimately ensured that when batches of vaccine were freeze-dried, the products should be stable and with qualified shape, then the freeze drying curve could be regarded as the standard. Among the freeze-drying curve, the formula E freeze-dried curve parameters are as follows:pre-freezing minimum temperature is-45 ℃, the vacuum degree of sublimation drying is 0.08 mbar, sublimation temperature is-15℃, desorption drying temperature is 26 ℃, all freezing process is about 43 h.After screening, the vaccine formulated with the formula E was stored respectively at 37℃, room temperature (20℃),2 to 8 ℃ by 10 days,2 months,1 year, after a defined interval, the EID50 of vaccine was determined, and conventional vaccine was set as a control. The results showed that, with time going, the virus titer of the vaccine continued to reduce. After being placed for 10 d at 37℃, its titer decreased from 10-4.32 to 10-3.38; in room temperature for 2 months, the titer dropped to 10-3.620 from 10-4.625, while the titer of conventional vaccine decreased from the 10-4. 75 to 10-3.52 in 1 week; in 2 to 8℃ for 12 months, the titer dropped down to 10-3.37 from 10-4.36, the titer was 10’382 when determined at the sixth months post-storage, while the conventional vaccine’s titer has been reduced from 10-4.45 to 10-3.44 in half of a year.Finally, the qualified vaccines were used in animal experiments, including the safety test, the immunity study and the antibody level monitoring. The chickens were divided into three groups:A (pre anti-aging vaccine), B (post anti-aging vaccine), C (PBS control),10 in each group. The vaccination dose of the safety test was 10 plumes each bird, and the survival rate of the three vaccination groups are all more than 80%. The M41 strain was used for challenge in the efficacy study. The efficacy of the two groups of A and B was both more than 80%. The diseased chickens and mock birds were dissected and the pathological sections of their trachea were made. The diseased chickens’trachea cilia shedding and cell necrosis were observed under an electron microscope, while the mock chickens’cilium was normal. Antibody monitoring results showed that the antibody kinetics of A, B groups were consistent, but that of A group was better. The highest level of antibody reached at 21-28 d post-vaccination in both groups.In summary, compared with the conventional vaccine, the efficacy for infectious bronchitis vaccine formulated with heat resistant protective agent in this study is better. This vaccine can be stored at 37℃ for 10 d, at room temperature of about 20 ℃ for 2 months, at 2 to 8℃ for more than 1 year, the preservation period is longer than that of the conventional vaccine. The heat-resistant protective agent can be helpful to improve the quality of the vaccine and to solve cold chain transportation. |
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