【作者】 王彦妮；

【导师】 田国彬；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2011， 硕士

【摘要】 H5亚型高致病性禽流感常常给养禽业造成巨大的经济损失,严重威胁世界各地的养禽业。禽流感的爆发能使肉鸡养殖业受到巨大的经济损失,这也与肉鸡的生长特点有直接关系。肉鸡生长周期短,成长迅速。这种快速增长方式使肉鸡长期处于应激反应状态,抵抗禽流感侵袭的能力也较弱,疫苗免疫预防肉鸡感染禽流感显得尤为重要。本文进行了肉鸡母源抗体保护时间、不同抗原浓度疫苗及不同佐剂疫苗的免疫效果、灭活疫苗与活疫苗配合使用效果等研究,并进行了疫苗免疫后的细胞免疫应答检测,以寻找对肉鸡比较理想的禽流感疫苗,为肉鸡禽流感疫苗防控提供科学依据。为观察肉鸡母源抗体的保护时间,本试验选择哈尔滨当地一批肉鸡,对肉鸡母源抗体的消长规律及不同时间攻毒保护进行了研究。结果显示肉鸡出生后1日龄时HI抗体滴度在4log2,之后一周内持续下降,到7日龄时已下降到3log2以下。肉鸡3日龄攻毒后死亡率为12/20,总排毒率为18/20:7日龄攻毒后死亡率为16/20,总排毒率为20/20;14日龄攻毒后,死亡率达到了19/20,总排毒率为20/20。肉鸡生长周期短且在上市之前必须具有一段休药期。因此,结合肉鸡母源抗体攻毒保护结果,确定免疫时间为7日龄左右。进行了H5亚型重组禽流感病毒(Re-5株)不同抗原浓缩倍数白油佐剂灭活疫苗对肉鸡免疫效果的比较。结果显示3倍、5倍抗原浓缩疫苗免疫肉鸡后1～6周HI抗体滴度高于非浓缩疫苗组。免疫后第6周时5倍抗原浓缩疫苗免疫组HI抗体滴度达到最高峰为6.3log2,3倍抗原浓缩疫苗免疫组为5.6log2,非浓缩疫苗免疫组仅为3.6log2。在免疫后2周攻毒试验中,浓缩疫苗免疫组肉鸡无死亡、无排毒。试验结果表明增加抗原含量有助于提高肉鸡对禽流感灭活疫苗的免疫应答。为筛选具有理想免疫效果的佐剂,本试验制备了4种不同佐剂的H5N1亚型(Re-5株)灭活疫苗,并对其进行了免疫肉鸡效果比较研究。试验结果显示,(Re-5株)Montanide ISA71VG油包水灭活疫苗免疫肉鸡后1～6周HI抗体滴度平稳上升,免疫后第6周HI抗体滴度达到4.7log2;攻毒后免疫组肉鸡无发病、无死亡、无排毒。从免疫鸡抗体检测和攻毒后的排毒情况来看,Montanide ISA71VG油包水佐剂,优于传统的白油佐剂以及Montanide ISA206VG佐剂和铝盐佐剂,具有良好的应用前景。本文对灭活疫苗与活疫苗联合免疫肉鸡后免疫效果进行了研究。试验结果显示:(Re-5株)Montanide ISA71VG佐剂油包水禽流感灭活疫苗及传统白油佐剂灭活疫苗与禽流感、新城疫重组二联活疫苗(rL-H5株)同时免疫或间隔1周免疫肉鸡,免疫后1～6周联合免疫的平均HI抗体滴度均高于相应疫苗单独免疫;而且在免疫后第1、2和3周攻毒时,疫苗联合免疫肉鸡组无死亡、无排毒。结果表明,禽流感灭活疫苗与活疫苗联合免疫,能够激发肉鸡理想的体液免疫应答。本研究可为灭活苗和活疫苗联合免疫程序提供参考。本文进行了已经应用或正在研究的H5亚型禽流感4种疫苗细胞免疫反应研究。结果显示,H5亚型重组禽流感(Re-5株)Montanide ISA71VG油包水灭活疫苗、(Re-5株)白油佐剂灭活疫苗、禽流感和新城疫重组二联活疫苗(rL-H5株)、DNA疫苗免疫肉鸡后,外周血及脾脏和胸腺中CD4+、CD8+、CD3+T淋巴细胞相对百分含量均明显高于未免疫组,且脾脏CD4+ T淋巴细胞呈阶段性上升状态;各时期SI值和巨噬细胞NO分泌量检测结果,免疫组均高于同期对照组(p<0.05),表明这4种疫苗均可以激发肉鸡产生较强的细胞免疫应答。更多还原

【Abstract】 H5 subtype Avian Influenza not only threats to poultry industry, but also causes huge economic losses all over the world in recent years.The growth characteristics of broilers are short growth cycle, rapid growth-up and most nutrients of feed turned into muscle growth. .So Broiler parts of the body are burdened and inside always in the state of stress reaction. As a result, broilers are poorer ability to resist avian flu. These cause more significant loss in broiler breeding industry in many countries and regions. The research on the time of maternal antibody against challenge, different cycles of concentration antigen of Inactivated oil-emulsion vaccines, immune effect of different vaccine adjuvant, effect of combined inactivated and live vaccine was studied and cellular immune response was detected, to find out satisfactory vaccines against Avian Influenza and provide scientific basis on prevention and control for broiler chickens Avian Influenza.Dynamic of maternal antibody and virus attack protection at different time was studied to observe the challenge time of maternal antibody by choosing a number of local broilers in Haerbin. The result is that HI antibody titers is 4log2 for one-day broiler and decline during the next week and is below 3log2 seven days later. The mortality is 12/20 and total shedding is 18/20. with 3-days vaccinated broilers, but 16/20 with7-days old and total shedding is 20/20. The mortality is up to 19/20 with 14-days vaccinated broilers.About 50 days old broiler will sell on the market, but they need some off-drug period for food safety. Based on experiment result, the broilers should be about 7 days old.By comparison of immune effect between different multiple cycles of concentration antigen of Inactivated oil-emulsion vaccines and (Re-5 Strain) Inactivated oil-emulsion vaccines, it shows that (Re-3 Strain) 3 cycles of concentration antigen of Inactivated oil-emulsion vaccines and (Re-5 Strain) 5 cycles of concentration antigen of Inactivated oil-emulsion vaccines have much better effect than (Re-5 Strain) Inactivated oil-emulsion vaccines. 0ne to six weeks later, HI antibody of different multiple cycles of concentration antigen is higher than none concentration antigen. HI antibody of 5 cycles of concentration antigen of inactivated oil-emulsion vaccines has maximum value 6.3log2, 3 cycles of concentration antigen of inactivated oil-emulsion vaccines 5.6log2 and none concentration antigen is 3.6log2. It turns out that increase antigenic content can improve immune reaction of avian influenza vaccine.4 (Re-5 Strain) different vaccine adjuvant inactivated vaccines have been developed to select desired adjuvant and compare the immune effect. (Re-5 Strain) Montanide ISA71VG Inactivated oil-emulsion vaccines Vaccinated one to six weeks later, HI antibody increases and up to 4.7log2 at the sixth week. Virus attack at two weeks later, no virus shedding, clinical signs, or deaths were observed. According to antibody level and detoxification, (Re-5 Strain) Montanide ISA71VG Inactivated oil-emulsion vaccines has better application prospect, compared with traditional Inactivated oil-emulsion vaccine、Montanide ISA206VG Inactivated oil-emulsion vaccine and aluminium adjuvant Inactivated oil-emulsion vaccine.Immune effect about vaccinated broilers was researched by using combined vaccines. It figures out that (Re-5 Strain) Montanide ISA71VG Inactivated oil-emulsion vaccines、Traditional (Re-5 Strain) Inactivated oil-emulsion vaccines combined with Avian Influenza Recombinant Newcastle Disease virus bivalent vaccine,Live(rL-H5 Strain) vaccinated at the same time or one-week interval, HI antibody are higher than none combined group after one to six weeks. Furthermore in virus attacking test at different time point, combined vaccines can provide completely immune protection of virus attack without detoxification or deaths. The results show that combined immune of inactivated vaccine against avain influenza and live vaccine triggers satisfactory humoral immune response. The research provides reference for immunization schedule of inactivated and live vaccines.In this paper cellular immune response was studied by 4 different kinds of H5 Subtype Vaccines against Avian Influenza. It figures out that while DNA vaccines , Avian Influenza Recombinant Newcastle Disease virus bivalent vaccine, Live(rL-H5 Strain),(Re-5 Strain)Montanide ISA71VG Inactivated oil-emulsion vaccines, (Re-5 Strain) Inactivated oil-emulsion vaccines immune chickens the relative percent contents of CD4+ and CD8+ subsets, CD3+ T lymphocytes in periphery blood, spleen and thymus of immunized groups are higher than the unvaccinated groups, and the relative percent contents of CD4+ T lymphocytes in spleen consistently increased after vaccination. Test result of SI-value and NO secretory volume of macrophages in the different stage display that immunized groups higher than the control groups (p<0.05), selected vaccine can stimulate strong cellular immune response with AA broiler.更多还原