论文标题:我国副鸡嗜血杆菌新血清型的分子病原学和免疫原性的比较研究
Comparative Studies on Molecular Characteristics and Immunogenicity of Haemophilus Paragallinarum Isolates of New Serovar
论文作者 卢冷轶
论文导师 赵德明；苗得园,论文学位 博士,论文专业 预防兽医
论文单位 中国农业大学,点击次数 637,论文页数 68页File Size5107k
2005-02-01论文网 http://www.lw23.com/lunwen_615942/ 副鸡嗜血杆菌;鸡传染性鼻炎;血凝素基因;免疫原性;三价疫苗
Haemophilus paragallinarum; infectious coryza; gene encoded hemagglutinin; antigenicity;trivaluent vaccine
2001年至2003年通过从不同地区的免疫失败鸡场分离到18株副鸡嗜血杆菌的血清型鉴定,有3株为B血清型,说明我国鸡传染性鼻炎的流行已经出现了新的特点。为解决临床上的问题,探讨免疫失败的原因,利用攻毒和血清HI抗体水平测定,观察不同A+C二价灭活疫苗二次免疫鸡群对B型分离株攻击的保护作用,结果表明:不同公司的疫苗的免疫效力有较大的差别,5个产品中有3个对A、C型保护较好,但不能对B型分离株的攻击提供有效的保护,且A、C型HI抗体阳性率、抗体滴度与对B型菌攻毒保护率无相关性,SD-1分离株可能是A型副鸡嗜血杆菌的变异株,其毒力已经有所加强。 鉴于B株流行和其他菌株毒力加强,为探求新菌株变异的分子病原学机制,本研究克隆了我国几个B型分离株和有代表性的A型株的血凝素基因,测定了其基因序列,从血凝素基因的水平上对国内的分离株进行了分析,并与已发表的标准株的序列进行了比较,结果表明,除Hp8株的基因与标准株极其类似外,SD-1、DL-1、TJ-1分离株的血凝素基因均多了一个氨基酸位点,与标准株的同源性也有所降低,进化树分析也完全属于一个新的进化支,说明我国的鸡传染性鼻炎的流行菌株在分子特性上出现了新特点,而多出的一个氨基酸位点,也极有可能是抗原性和菌株变化的重要原因。 为更好地控制B型菌的感染,为三价疫苗的研制提供依据,通过攻毒保护试验和HI抗体测定对我国不同地区的B型副鸡嗜血杆菌分离株进行了免疫原性的比较。分析结果表明:不同地区的分离株免疫原性存在一定的差异,在目前得到的分离株中,TJ-1免疫鸡对TJ-1和DL-1株的保护作用可达到90%和91.7%,对BJ-1株的保护率为63.6%,具有相对较好的保护作用,目前可以作为疫苗株使用。根据免疫原性的比较结果,选择抗原性较好的B型分离株TJ-1参与制备了鸡传染性鼻炎三价油乳剂灭活疫苗,并应用3批鸡传染性鼻炎三价油乳剂灭活疫苗进行了安全性和免疫效力检验,结果发现,超剂量注射疫苗鸡只食欲、精神未见异常,2周后观察,注射部位从外部观察未发现明显异常和病变。用该疫苗免疫后的鸡只对A型强致病力菌株的攻击保护率可达100%,对B型菌株的攻击保护率为94.4%,对C型高致病力菌株的攻击保护率为93.3%。采用阻断ELISA、HI和SPA对鸡群的抗体水平的评估表明该疫苗可激发产生较高的抗体水平。所有结果均表明,该疫苗具有良好的安全性和近期免疫效力。
About 18 Haemophilus paragallinarum (Hpg) strains were isolated and identified in different sites, there are 3 B serovar strains in them, indicating the new epidemiological characteristics of infectious coryza in China. In order to resolve the field problems, analysis the reasons for immunity failure, the protection ability against B serovar of chickens immunized with different A+C vaccine were studied by challenging and antibody test, the results showed that, vaccines from different producer got different efficacy, 3 products in 5 gave good protection to challenge with A or C Hpg, but failed to gave good protection against challenge with B strain, and there is no obvious correlation between A or C antibody titer and protection rate for serovar B. Strain SD-1 is a A serovar variant with enhanced pathogenicity.Regarding the infection of serovar B and the virulent variety of A serovar, also to analysis the molecular mechanism of the variations among isolates, the genes encoded Hpg hemagglutinin antigen were coloned, sequenced and compared with the published sequences for reference strains, the results demonstrated that, except for the similarity between Hp8 and 0221, SD-1, DL-1 and TJ-1 containing 3 accessorial bp, the homogencity between the reference strains and native isolates was also decreased, the phylogenetic tree indicated these 3 isolates belonged to the same branch and there were some new molecular characteristics in the strain prevalent in China, the accessorial 3 bp was most likely the reason to the alteration for antigenicity and pathogenicity.In order to prevent the infection of serovar B and get reference for the development of trivaluent vaccine, the antigenicities of the B serovars were compared with challenging and antibody test, the results showed that there are some different among the strains from different sites. TJ-1 immunized chicken achieved an 90%, 91.4% and 63.6% protective rates for the challenge against TJ-1, SD-1 and BJ-1 respectively, so TJ-1 could be the most suitable vaccine strain at this time. According to results for antigenic comparation, 3 lots of trivaluent vaccine were prepared (TJ-1 strain was used as B serovar vaccine strain) and their safety and efficacy were also tested. There is no obvious change in appetite and spirit of the chickens injected with 2.0 ml vaccine, there is also no significant pathological damage being observed in vitro after 2 weeks postinjection, the vaccinated chickens achieved 100%, 94.4% and 93.3% protective rates to the challenge against A, B, C high virulent strain. We also detected high level of antibodies to B-ELISA, SPA and HI in the sera from vaccinated chickens, all the results demonstrated the good safety and efficacy of the the developed trivaluent vaccine.
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