【作者】 祝明；

【导师】 王健；

【作者基本信息】 西南大学 ， 临床兽医学， 2009， 硕士

【摘要】 禽流感（Avian Influenza,AI）是由禽流感病毒（Avian Influenza Virus,AIV）引起的一种禽类急性传染病。自1997年至今,不仅屡次造成各国禽业的巨大损失,而且频频突破种间屏障,致使全球有近200人感染,100多人死亡。随着不断研究发现,水禽不仅已成为禽流感病毒高度易感禽类,而且是禽流感病毒的巨大贮存库和重要传染源。目前,我国防控HPAI的有效途径是注射油乳剂灭活疫苗。但该疫苗仍有诸多缺陷。一是免疫后机体形成有效抗体滴度的间隙期较长,不能满足紧急情况下快速建立免疫保护的要求;二是油佐剂刺激性大、难吸收,在肉鸭宰杀销售之时仍有部分油佐剂残留于机体,严重影响产品质量。因此,寻求油佐剂替代佐剂,填补现行禽流感灭活油苗的缺陷已成为迫切需要。随着纳米技术的广泛应用,已有研究显示无机纳米铝佐剂可能最具曙光。但是纳米氢氧化铝佐剂辅佐的禽流感疫苗对肉鸭的特异性免疫、非特异性免疫、免疫器官发育和粘膜免疫等方面效果的影响还未见报道。针对以上问题,本课题用纳米氢氧化铝佐剂物理吸附禽流感H5N1灭活抗原,制作了纳米Al（OH）₃辅佐AI H₅N₁灭活疫苗,并以肉鸭为实验动物,旨在研究:①纳米Al（OH）₃辅佐AI H₅N₁灭活疫苗对肉鸭特异性免疫的影响;②纳米Al（OH）₃辅佐AI H₅N₁灭活疫苗对肉鸭非特异性免疫的影响;③纳米Al（OH）₃辅佐AI H₅N₁灭活疫苗对肉鸭免疫器官的影响;④纳米Al（OH）₃辅佐AI H₅N₁灭活疫苗对肉鸭黏膜免疫的影响。本研究将为纳米Al（OH）₃辅佐AI H₅N₁灭活疫苗在肉禽养殖中的实际应用提供理论依据,同时为肉鸭的禽流感防治工作提供理论参考。本研究分为以下几部分:1.纳米Al（OH）₃辅佐AI H₅N₁灭活疫苗对肉鸭特异性免疫的影响研究将108只雏鸭随机分入4组,各组受试鸭腿部肌肉注射免疫物0.5ml/只。免疫后第3、5、7、10、14、21、28、35天按血凝抑制（HI）试验方法检测其AIV抗体滴度。免疫后第7天,第21天,第35天,测定E玫瑰花环形成率和淋巴细胞增殖活性。结果显示:纳米铝佐剂疫苗能显著提高机体抗体应答的速度,产生有效保护抗体时间较油佐剂疫苗和常规铝佐剂疫苗提前3天和7天,抗体峰值达到时间较油佐剂疫苗提前14天,但抗体持续时间不如油苗持久;在玫瑰花环形成率和淋巴细胞增殖试验中,纳米铝佐剂组与其他三组没有显著差异（P＞0.05）。结果表明:纳米氢氧化铝作为H5N1亚型禽流感疫苗的免疫佐剂,可以更早的刺激动物产生有效保护抗体,并在肉鸭的早期生长阶段,对细胞免疫具有一定的刺激作用,但并不能激发肉鸭明显的细胞免疫。2.纳米Al（OH）₃辅佐AI H₅N₁灭活疫苗对肉鸭非特异性免疫的影响研究将68只雏鸭随机分入4组。在免疫后第7天,第21天,第35天,进行碳廓清试验和溶菌酶含量测定。结果显示:免疫后第7天,纳米组的碳廓清指数相对较高,与常规铝组相比差异显著（P＜0.05）:免疫后第21天和第35天,纳米组碳廓清指数与其他三组差异不显著（P＞0.05）。免疫后第7天,纳米组的溶菌酶含量较高,与油乳剂组、常规铝组和生理盐水组均差异显著（P＜0.05）;免疫后第21天和第35天,纳米组碳廓清指数与其他三组差异不显著（P＞0.05）。结果表明:在肉鸭生长的早期阶段,纳米铝佐剂禽流感疫苗对肉鸭血清中溶菌酶含量和巨噬细胞的吞噬能力都有一定的提高作用,说明纳米氢氧化铝作为H5N1亚型禽流感疫苗的免疫佐剂,可以提高早期肉鸭巨噬细胞的活性,增强非特异性免疫的能力。3.纳米Al（OH）₃辅佐AI H₅N₁灭活疫苗对肉鸭免疫器官的影响研究将68只雏鸭随机分入4组。免疫后第7天,第21天,第35天,将鸭称重后处死,接着取出胸腺、脾脏和法氏囊称重,计算脏/体比值。结果显示:免疫后第7天,法氏囊指数相对于油乳剂组、常规铝佐剂组和生理盐水组差异显著（P＜0.05）。胸腺指数和脾脏指数相对于其他三组差异不显著（P＞0.05）。免疫后第21天和第35天,三种免疫器官的指数均差异不差异（P＞0.05）。结果表明:纳米疫苗在一定程度上能促进肉鸭免疫器官的发育特别是法氏囊的发育从而在一定程度上增强肉鸭的免疫力。4.纳米Al（OH）3辅佐AI H5N1灭活疫苗对肉鸭粘膜免疫系统的影响研究将75只雏鸭随机分入3组。至14日龄各组受试鸭鼻腔接种免疫物0.5ml/只。免疫后第7、14、21、28、35天,按照血凝（HA）和血凝抑制（HI）试验技术对不同时期的粘液样品进行AIV抗体滴度抗体的检测。结果显示:免疫后第7天,纳米组抗体水平极显著高于灭活抗原组和对照组（P＜0.01）;免疫后第14天,纳米组和灭活抗原组的抗体效价分别达到高峰值,纳米组抗体水平极显著高于灭活抗原组和对照组（P＜0.01）。结果表明:鼻腔途径接种纳米Al（OH）₃辅佐AI H₅N₁灭活疫苗能够诱导肉鸭产生一定的黏膜免疫反应,有效加强家禽局部免疫力。更多还原

【Abstract】 Avian Influenza is an acute infectious disease caused by Avian Influenza Virus.It not only caused great damage in poultry enterprise repeatedly but also breakthrough interspecific barrier and left 200 dead more than 100 infected in the world.Research continues to show that waterfowl not only turned easily affected by AIV but also became a huge store and the source of AIV.At present, vaccine with whole inactivated virus and oil adjuvant is a important measures for the prevention and control of HPAI outbreaks and spread.But this vaccine has some deficits.Firstly,there is a long vacant period before forming effective antibody.Secondly,the excitation of oil adjuvant has a strong impact on quality of product.Consequently,it is imperative to seek a new adjuvant to replace oil adjuvant.The studies indicated that nanoparticulated aluminum hydroxide adjuvant has the highest hopes with the applications of Nanotechnology.however,the effect of specific immunity、nonspecific immunity、growing of immune organ and respiratory tract immunity of avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant(NAHA) in duck is unknown.To the above question,in this experiment ducks were immunized with avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant(NAHA) to research:①the effect of avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant(NAHA) on specif immunity in duck;②the effect of avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant(NAHA) on nonspecific immunity in duck;③the effect of avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant(NAHA) on growing of immune organ in duck;④the effect of avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant(NAHA) on growing on respiratory tract immunity in duck.The purpose of this study was to provide theoretic foundation for the practical applications of avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant and offer some references for the prevention of avian influenza.This experiment can be divided into the following parts:1.The effect of avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant(NAHA) on specif immunity in duck.108 ducklings whose antibodies were 0 in 10 days old were randomly allotted in 4 different treatments and immuned by intramuscular injection in leg in does of 0.5ml.The level of antibody was detected by HA and HI at 3、5、7、10、14、21、28、35 days after immune..The lymphocytes was determined on EAC and MTT at 7、21、35 days after immune.The results showed that:NAHA can significantly enhance the speed of the antibody response of body,the time emerging effective antibody of NAHA group advanced by 3 and 7 days compared to OE and AHA group respectively, the time to reach the highest antibody of NAHA group is less than OE 14 days.However,the duration of antibody of NAHA group is not durable as the OE.There is no obvious difference in Rosette forming rate(EAC) and transformation rate of lymphocytes(MTT) between NAHA group and other three groups(P>0.05).The result indicate that:Nanometer-aluminium hydroxide adjuvant could stimulate animals to produced effective protection antibody earlier.And it can stimulate Cellular Immunity in the early growth stage of duck.But it does not significantly provocative the cellular immune of the duck.2.The effect of avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant(NAHA) on nonspecific immunity in duck.68 ducklings were randomly allotted in 4 different treatments,the content of serum lysozyme and the cytophagic index of macrophage was determined on EAC and MTT at 7、21、35 days after immune.The results showed that:the cytophagic index of macrophage of NAHA group is obvious different compared with Aluminum Hydroxide Adjuvant group at 7 days after immune(P<0.05 )and not obvious different compared with other three groups at 21、35 days after immune.(P>0.05).;The content of serum lysozyme of NAHA group is obvious different compared with other three groups at 7 days after immune(P<0.05) and not obvious different compared with other three groups at 21、35 days after immune.(P > 0.05);The result indicate that: Nanometer-aluminium hydroxide adjuvant can not only increase the content of duck serum lysozyme and enhance the activity of macrophage in the early stage of the duck but also improve the capacity of non-specific immune. 3.The effect of avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant(NAHA) on growing of immune organ in duck.68 ducklings were randomly allotted in 4 different treatments.Ducks were killed after the body weighted,then Remove the thymus,spleen and bursa of fabricius and weighed them,then calculated the ratio of internal organs and body weight.The results showed that:The index of bursa of fabricius is obvious different compared with other three groups at 7 days after immune(P<0.05),The indexes of thymus and spleen are not obvious different compared with other three groups at 7 days after immune(P > 0.05);The indexes of these immune organ are not obvious different compared with other three groups at 21、35 days after immune(P>0.05).The result indicate that:to some extent, Nanometer-aluminium hydroxide adjuvant can promote the development of immune organs duck, especially the bursa of fabricius,base on this reason,it had the effects on enhancing immunity of duck.4.The effect of avian H5N1 influenza virus vaccine and nanoparticulated aluminum hydroxide adjuvant(NAHA) on growing on respiratory tract immunity in duck.75 ducklings were randomly allotted in 3 different treatments.They were immunized via intranasal in 14 days in does of 0.5ml.Venous samplings were collected at3、5、7、10、14、21、28、35 days after immune.The level of antibody was detected by HA and HI.The results showed that:the antibody level of NAHA is significant compared to H5 and control group respectively at 7 days after immune(P<0.01);The levels of antibody of NAHA group and OE group reached a peak value respectively at 14 days after immune,the antibody level of NAHA is significant compared to H5 and control group respectively at 14 days after immune(P<0.01).The result indicate that:duck were immunized via intranasal with inactivated of avian H5N1 influenza virus vaccine and adjuvant nanoparticulated aluminum hydroxide could induce mucosal immune reaction,improve the local immunity effectively.更多还原