【作者】 黄志永；

【导师】 韦平；

【作者基本信息】 广西大学 ， 预防兽医学， 2008， 硕士

【摘要】 传染性法氏囊病（infectious bursal disease,IBD）是由传染性法氏囊病病毒（infectious bursal disease virus,IBDV）引起的主要危害幼龄鸡的一种急性、高度接触性的传染病,除引起发病、死亡等直接的损失外,还可导致感染鸡产生免疫抑制,致使机体免疫应答降低和对其它疾病的易感性增高,造成更大的损失。基因突变所引起的IBDV超强毒株（very virulent IBDV,vvIBDV）和不同血清亚型的出现,是造成目前现有商品疫苗免疫效力不高或免疫失败的主要原因之一。因此,及时分离并筛选具有代表性的地方流行毒株作为研制疫苗的候选毒株,并对其免疫原性和交叉免疫保护性进行试验就成为一项十分有意义而且非常必需的工作。在之前的研究中,我们在对2000～2006年间分别来自广西、江苏、浙江、安徽、海南5个省的23个IBDV分离毒株的主要保护性抗原蛋白VP2基因的高变区序列进行比较分析后发现,分离株可明显分为2群:第1群为属于经典毒株（classical IBDV,cIBDV）的10个弱毒株及2个中等偏强毒力的分离株,第2群为属于vvIBDV的11个分离株。本课题选取分离自广西的属第2群的vvIBDV毒株BH11、TSC-2（9）和属第1群的中等偏强毒力株040124、YL052作为代表,与目前在生产上使用最广泛的商品疫苗株B₈₇（in）和FW2512一起,进行了毒株血清亚型和致病力鉴定、毒株间交叉免疫保护等研究,目的是筛选出致病性强、免疫原性和免疫交叉保护性好的毒株作为研制疫苗的种毒。首先,通过应用免疫兔子制备的抗IBDV高免血清,在鸡胚成纤维细胞（CEF）上对四个流行代表株040124、BH11、TSC-2（9）、YL052和2个疫苗株B₈₇（in）、FW2512进行交叉中和试验。结果显示,6个毒株可分成2个不同的血清亚型:BH11为一个亚型,YL052、TSC-2（9）、040124与疫苗株B₈₇（in）和FW2512为另一个亚型;通过聚类分析法分析了各亚型毒株之间的亲缘关性存在一定的差异。第二,用四个分离株分别人工感染4周龄非免疫鸡,以比较和确定它们的致病性。实验从致死率、免疫器官指数（immune organs index,IOI）及法氏囊显微病理损伤评分分别进行评价。结果显示四个毒株对实验鸡的发病率均为100%;在各实验组与对照组的IOI比较中差异均显著（p＜0.05）,表明它们均具有一定程度的致病性,但各毒株间的毒力差异较大,040124毒株的致死率（40%）和法氏囊显微病理损伤评分（5分）均为最高。第三,通过制备四个分离株的单价灭活油乳剂疫苗（OEV）、多价灭活OEV（040124、TSC-2（9）、BH11）以及参考强毒株CJ801-BKF的OEV,分别进行商品疫苗毒株B87（in）、FW2512对4个分离株的免疫保护试验、各分离株间的免疫交叉保护试验、多价灭活苗不同免疫剂量及其与CJ801-BKF株灭活苗的免疫保护对比试验。试验分别从攻毒后各毒株疫苗免疫组鸡的IOI、免疫保护指数（immune protection index,IPI）进行评价。结果显示,B₈₇（in）和FW2512疫苗免疫组对四个分离株YL052、040124、BH11、TSC-2（9）、的IPI分别为80%、60%、70%、70%和70%、60%、70%、80%,表明这两个商品疫苗不能对流行毒株提供完全的保护;四个单价OEV对同源毒株均可产生100%保护,040124的OEV对BH11、TSC-2（9）、YL052的IPI分别为80%、80%、90%,交叉免疫保护性最好,其它三个毒株间的IPI则在40%～70%;在毒株免疫组与未免疫组的IOI比较观察中发现,分离株TSC-2（9）和YL052免疫组与未免疫组的差异显著（P＜0.05）,而分离株040124和BH11免疫组与未免疫组的IOI差异则不显著（P＞0.05）,综合两项指标的结果,证明毒株040124株的免疫原性最好;多价OEV三个免疫剂量的的IPI都在80%以上,最高的达100%,各免疫剂量的比较中,1mL的免疫组IPI显著高于0.25mL的免疫组（P＜0.05）,而1mL免疫组与0.5mL免疫组差异不显著（P＞0.05）,可以确定0.5mL为多价OEV的最佳免疫剂量;应用最佳免疫剂量的多价OEV所进行的免疫保护试验中,发现它对四个分离株的IPI达90%以上,而CJ801-BKF株灭活OEV对各BH11、TSC-2（9）、YL052、040124的IPI分别只有65%、75%、60%、50%;在对多价OEV、4个单价OEV和CJ801-BKF株OEV的IPI比较中,多价OEV的免疫效力显著高于YL052、TSC-2（9）株和CJ801-BKF株的OEV（P＜0.05）,各毒株OEV之间及其与CJ801-BKF株OEV以及多价OEV与毒株040124、BH11株OEV之间的差异均不显著（P＞0.05）。综合以上结果,证明本研究制备的多价灭活OEV的免疫效力最高,可适用于本地IBD的防制。本课题研究的结果表明,广西存在不同的IBDV血清亚型的流行,商品疫苗B₈₇（in）和FW2512已不能对流行毒株提供完全的保护,研发多价油乳剂灭活疫苗是本地防控IBD一种有效的策略。更多还原

【Abstract】 Infectious bursal disease virus(IBDV)is the etiological agent of infectious bursal disease(IBD),which is an acute,highly contagious disease,and either causes significant losses to the poultry industries by causing high mortality or even more infections as a consequence of immunosuppression induced by IBDV infection in young chickens,which decreases the host’s immune response and increases susceptibility to other diseases.Partially protection or immune failure in the vaccinated flocks dued to the emergence of new subtypes and the very virulent strains of IBDV(vvIBDV)was one of the consequences of gene mutations of IBDV.It is important and essential to isolate the epidemic isolates and screening for the candidate strain or strains by testing of immunogenicity and cross immunity protection.In our previous research,a phylogenetic tree based on the nucleotide sequence of high variance region of VP2 gene of 23 IBDV strains was made which were isolated from Guangxi,Jiangsu,Zhejiang,Anhui and Hainan provinces during the years of 2000 to 2006,and it indicated that 23 isolates were classified into 2 clusters:the first which belonged to cIBDV includes 10 attenuated isolates and 2 intermediate virulence isolates YL052 and 040124,the second which belonged to vvIBDV includes 11 isolates.In the study 4 isolates included 040124 and YL052,BH11 and TSC-2(9)in cluster 2,and 2 vaccine strains B87(in)and FW2512 which were widely used in the industry,were evaluated on serum subtype,pathogenicity,immunogenicity and cross protective immunity among the viruses for the purpose of screening vaccine candidate strain or strains based on high virulence,good immunogenicity and cross protective immunity.Firstly,hyper-immuned rabbit antisera against the four isolates and the two vaccine viruses were used in a virus neutralization(VN)test carried out on chicken embryo fibroblast(CEF)against the heterogeneous and homogeneous strains.Relatedness values of each virus calculated based on the cross-neutralization antibody titer showed that 2 subtypes were distinguished among the 6 viruses tested:BH11 belongs to a subtype,while TSC-2(9),040124, YL052,B87(in)and FW2512 belong to another subtype.Phylogenetic analysis on the viruses of different subtypes indicated that there was divergence in antigenecity among the field isolates and between the isolates and the vaccine viruses.Secondly,four isolates were used to infect non-vaccined 4-week-old chickens to compare and determine their pathogenicity by analyzing the lethality,immune organ index and the micro-pathology lesions score of bursa.The results showed that there was significant differences of immune organ index(IOI)between the infected groups and the un-infected control group(P＜0.05)even all the four isolates could induced morbitities of 100%,indicated that there is difference among the isolates on pathogenicity with the highest lethality(40%)and points (5)of micro-pathology lesions score of bursal.Thirdly,immune protection test about vaccine strains B87(in)and FW2512 against the 4 isolates,and cross-protective immunity tests on the viruses,and protection contrast test of multivalent inactivated oil-emulsion vaccine(OEV) with different immune dosage,and protection contrast test of multivalent OEV and reference velogenic strain CJ801-BKF were conducted by vaccinating the birds with OEV of the 4 isolates,multivalent OEV,the immune protection index(IPI)and immune organs index(IOI)were evaluated respectively post-challenge.The results showed that the protections of B87(in)and FW2512 against YL052,040124,BH11,and TSC-2(9)were 80%,60%,70%,70%and 70%,60%,70%,80%respectively,it indicated that both two commonly used vaccine viruses can’t provide full protection against the field isolates. Protections of 100%were observed on the birds vaccinated with OEV of the 4 isolates against the homogeneous viruses,and the protections of OEV of 040124 against isolates BH11,TSC-2(9)and YL052 were the best by 80%,80%and 90%respectively,while those of the other three isolates were divergent (40%-70%).Birds vaccinated with OEVs of isolates TSC-2(9)and YL052 had significant difference of IOI with the un-infected control birds(P＜0.05),while the birds vaccinated with OEVs of isolates 040124 and BH11 had no significant difference of IOI with the un-infected control birds(P＞0.05).It is concluded that the immunogenicity of 040124 was the best among the isolates.IPI of the multivalent OEV with 3 different immune dosage is 80%～100%,dosage 1mL and 0.5mL had significant higher IPI than that of dosage 0.25 mE(P＜0.05), while dosage 1mL and 0.5mL had no significant difference on IPI(P＞0.05),it indicated that the optimal immune dosage of multivalent OEV is 0.5mL/bird. The application of the multivalent OEV with the optimal immune dosage showed that the IPI against the 4 isolates is about 90%,while the IPI of CJ801-BKF against BH11,TSC-2(9),YL052 and 040124 were 65%,75%,60% and 50%respectively;In the comparison among the multivalent OEV,OEVs of the 4 isolates and CJ801-BKF,indicated that IPI of multivalent OEV is significant higher than that of OEVs of YL052,TSC-2(9)and CJ801-BKF(P＜0.05),while there was no significant difference on IPI among the 4 isolates, between them and CJ801-BKF,between multivalent OEV and 040124, BH11respectively(P＞0.05).It is concluded that the immune efficacy of multivalent OEV was the best among all the OEVs tested,which may be a good vaccine for the application in Guangxi.The results of study demonstrated that there were different subtypes of IBDV prevalent in Guangxi.Vaccine strains B87(in)and FW2512 can’t provide full protection against the epidemic isolates,and development of multivalent OEV is a effective strategy for prevention and control of IBD in Guangxi.更多还原