重组鸡白细胞介素-2对禽流感灭活疫苗免疫增强作用的研究

为研究重组鸡白细胞介素-2(IL-2)对H5N1亚型禽流感灭活苗的免疫效果,将重组IL-2蛋白和禽流感灭活疫苗联合免疫14日龄SPF鸡,免疫后每周测定一次血清抗体效价﹑脾脏T淋巴细胞亚群,最后计算攻毒保护率。试验结果表明:重组IL-2蛋白具有增强禽流感疫苗免疫效果的作用,提高了血清中的抗体水平﹑增加了脾脏T淋巴细胞CD4+﹑CD8+亚群数量。     

缓释复方免疫增强剂对鸡传染性法氏囊病疫苗免疫增强效果研究

将100只20日龄海兰褐蛋雏鸡随机分成试验组和对照组．分别肌肉注射IBDB87疫苗0．5mL／只,同时给试验组雏鸡投服缓释复方免疫增强剂l粒／只．对照组鸡不投服．在免疫前和免疫后10、20、30d,每组随机采血．测定鸡IBD抗体阳性率、T淋巴细胞ERFC形成率、脾脏和法氏囊指数。结果表明．免疫后不同时期鸡IBD抗体阳性率、T淋巴细胞ERFC形成率、睥脏和法氏囊指数试验组显著或极显著高于对照组．说明缓释复方免疫增强剂可以提高鸡体液免疫功能和细胞免疫功能．促进免疫器官生长发育．     

重组鸡IL-2作为传染性法氏囊病疫苗免疫增强剂对鸡细胞免疫水平的影响

传染性法氏囊病是危害养禽业的一种最为重要的疾病之一，经常造成鸡只的死亡，耐过鸡免疫抑制，以后疫苗接种免疫失败和产蛋率下降，造成严重的经济损失。传染性法氏囊病活苗免疫效果虽然较好，但保护期短，易引发感染和免疫抑制；灭活苗较安全，保护期长，但效果较差，应寻找一种免疫增强剂，     

缓释复方免疫增强剂对鸡传染性法氏囊病疫苗的免疫增强试验

鸡传染性法氏囊病是由传染性法氏囊病病毒引起的一种急性、高度接触性传染病,目前防治该病主要应用弱毒苗和灭活苗,但由于病毒变异和超强毒株的出现、病原体混合感染的增加以及其它疾病引起的免疫抑制等因素,常造成鸡传染性法氏囊疫苗免疫效果不佳甚至失败,给养鸡业造成了巨大的经济损失。     

重组鸡白细胞介素18(rChIL-18)对MDV感染SPF鸡细胞免疫的影响

用马立克氏病病毒（MDV）感染5日龄SPF鸡后,取21日龄和35日龄鸡的淋巴细胞,运用3H-TdR掺入法检测T淋巴细胞对ConA和重组鸡白细胞介素18（rChIL-18）的反应,并用MTT法检测NK细胞和CTL对MD肿瘤细胞系CU147的杀伤活性及rChIL-18和IFN-γ对它们的作用。结果显示,SPF鸡感染MDV后淋转水平显著下降,NK细胞、CTL杀伤活性在感染后21日龄时升高,而在35日龄时NK细胞杀伤活性显著下降。rChIL-18对对照组和感染组SPF鸡的淋转水平和杀伤活性均有提高作用,同样IFN-γ也具有提高NK细胞和CTL杀伤活性的作用。     

缓释复方免疫增强剂对鸡传染性法氏囊病疫苗免疫增强效果研究

鸡传染性法氏囊病（IBD）是鸡的一种急性、高度接触性传染病,防治主要应用弱毒苗和灭活苗,但由于病毒变异和超强毒株出现,病原体混合感染,其它疾病引起免疫抑制,应激因素的刺激等,造成鸡IBD疫苗免疫效果不佳甚至失败。如何提高IBD疫苗的免疫效果已成为研究的热点。陆钢等（1995）在雏鸡免疫ND和IBD疫苗前后,以0．2％蜂花粉拌料,肌注芦荟提取液0．1mL／只,在二免后1个月时,ND和IBD效价较对照组提高1个滴度,差异极显著。     

重组鸡IL-2对禽流感灭活苗免疫增强作用试验

使用重组鸡白介素-2作为免疫佐剂,检测chIL-2对禽流感病毒H9亚型灭活疫苗的免疫增强作用。将1日龄的AA肉雏鸡,随机分为7组,每组10只,采用胸部肌肉注射方式进行免疫,对照组仅接种H9亚型禽流感灭活疫苗,试验组分别同时接种H9亚型禽流感灭活疫苗O．5mL和不同剂量（0．01mg-1mg）的鸡IL-2。每7d静脉采血,采用血凝和血凝抑制方法检测机体中抗体水平变化情况。结果表明,接种重组鸡IL-2的试验组动物能在2月内保持高抗体水平,且在免疫后第10周,其平均抗体水平仍高于疫苗对照组3．6个～4．5个滴度,表明了重组鸡IL-2蛋白对H9亚型禽流感灭活疫苗具有免疫增强作用。     

缓释复方免疫增强剂对鸡传染性法氏囊病疫苗增强效果研究

100羽20日龄海兰褐蛋雏鸡随机均分为试验组和对照组,均肌肉注射IBDB87疫苗0.5mL/羽,同时给试验组雏鸡投服缓释复方免疫增强剂1粒/羽。在免疫前、免疫后10、20和30d,每组随机采血20羽,测定IBD抗体阳性率、T淋巴细胞ERFC形成率、脾脏和法氏囊指数。结果2组鸡上述测定指标具有相同的消涨趋势,且试验组显著或极显著高于对照组,表明缓释复方免疫增强剂可提高鸡体液和细胞免疫功能,促进免疫器官生长发育。     

鸡新城疫和传染性法氏囊病灭活疫苗抗原量对鸡细胞免疫的影响

将不同抗原含量的鸡新城疫和传染性法氏囊病油乳剂灭活疫苗分别免疫１７日龄雏鸡，测定了疫苗抗原含量与细胞免疫应答之间的关系。经Ｔ淋巴细胞转化试验结果表明，两种疫苗的抗原含量－细胞免疫应答曲线是一致的，随着抗原量的增加，活化的Ｔ淋巴细胞数量也相应增多，但是细胞免疫应答的可测时间是暂时的，于免疫７天达高峰，１７天后下降接近正常水平。     

重组鸡α-干扰素对鸡传染性法氏囊炎的治疗试验

重组鸡α-干扰素是一种抗病毒蛋白,具有广谱的抗病毒活性,对多种病毒有抑制作用。为了验证其治疗效果,在本试验中,我们使用法氏囊标准强毒BC6／85对易感日龄的SPF鸡进行人工感染与治疗试验,在攻毒后临床症状出现时使用10万单位、1万单位和0．1万单位三个剂量的重组鸡α-干扰素进行了治疗试验,同时使用法氏囊卵黄抗体做为对照药品,进行对比试验。结果表明,与攻毒对照组相比较,重组鸡α-干扰素能够显著降低试验鸡的发病数量,减轻其临床发病症状和法氏囊病变,并且在生产性能上具有较好的增重作用,其中以1万单位的中剂量重组鸡α-干扰素效果最为显著。     

The role of T cells in protection by an inactivated infectious bursal disease virus vaccine

The current belief is that the humoral immune response plays the principal role in defense against virulent infectious bursal disease virus (IBDV). In this study we used a model, in which chickens were compromised in functional T cells by neonatal thymectomy and Cyclosporin A (TxCsA) treatment, to demonstrate the role of T cells in protective immunity against IBDV. We demonstrated that T cells were necessary to achieve full protection against virulent IBDV. When T cell compromised TxCsA-treated chickens were vaccinated with an inactivated IBDV (iIBDV) vaccine, 91% were not protected against IBDV challenge in comparison to T cell-intact chickens, which had a protection rate of 91%. The iIBDV vaccine induced virus neutralizing (VN) and ELISA antibodies, respectively, in 65 and 5% of TxCsA-treated, and in 100 and 58% of T cell-intact birds. These observations provide evidence that the stimulation of T helper cells is needed for the production of protective antibody levels in iIBDV-vaccinated chickens. Passive administration of VN anti-IBDV antibodies inducing a circulating antibody level of log(2)8 in chickens revealed that the levels of antibodies that protected T cell-intact chickens against virulent IBDV challenge were not protective for TxCsA chickens. These results indicated that antibody alone was not adequate in inducing protection against IBDV in chickens and that T cell-involvement was critical for protection. We propose that the inability of iIBDV to protect TxCsA chickens was due to compromised T cell immunity, functional T helper cells and most likely also cytotoxic T cells are needed in iIBDV vaccine protection.     

Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens

A recombinant fowlpox virus (rFPV/VP2) expressing infectious bursal diseases virus (IBDV) VP2 gene has been constructed. After purification and identification of rFPV/VP2, the adjuvant activity of the recombinant chicken IL-12 (rchIL-12), synthesized by our previous construct of rFPV/chIL-12, in rFPV/VP2-expressed rVP2 antigen was assessed in one-week-old specific-pathogen free chickens. The results indicated that rchIL-12 alone or rchIL-12 plus mineral oil (MO) co-administered with rVP2 antigen significantly enhanced the production of serum neutralization (SN) antibody against IBDV, compared to those with MO alone. The SN titers in groups receiving rVP2 antigen with MO alone were more inconsistent after vaccination. On the other hand, rchIL-12 significantly stimulated IFN-γ production in serum and in splenocyte cultured supernatant, suggesting that rchIL-12 alone or plus MO significantly induced a cell-mediated immune response. Finally, bursal lesion protection from very virulent IBDV (vvIBDV) challenge in chickens receiving rVP2 antigen with rchIL-12 alone or plus MO was much more effective than that with MO alone at two weeks after boosting. Taken together, rchIL-12 alone augmented in vivo the induction of a primary and also a secondary SN antibody production and a cell-mediated immunity against IBDV rVP2 antigen, which conferred the enhancement of bursal lesion protective efficacy from vvIBDV challenge. These data indicated that a potential for chIL-12 as immunoadjuvant for chicken vaccine development such as IBDV rVP2 antigen.     

鸡传染性贫血病毒对鸡传染性囊病活疫苗(Gt株)免疫效果的影响

鸡传染性贫血(CIA)是由鸡传染性贫血病毒(CAV)引起的一种以再生障碍性贫血和淋巴器官组织萎缩为主要特征的免疫抑制病.崔现兰(1992)首次于我国东北地区分离到该病毒[1],目前在我国许多地方都有该病毒的报道.CAV侵入雏鸡体内后,主要作用于骨髓和胸腺等淋巴器官,导致感染鸡出现再生障碍性贫血及胸腺等淋巴器官严重萎缩,产生免疫抑制,对禽病疫苗的免疫应答机能降低,甚至丧失.为研究鸡传染性囊病病毒对鸡传染性囊病活疫苗(Gt株)免疫效果的影响,本试验采用人工感染CAV的方法研究CAV感染对鸡传染性囊病活疫苗(Gt株)免疫效果的影响.     

表达抗鸡传染性法氏囊病病毒重组鸡源抗体CHO细胞的悬浮驯化

为将贴壁生长的表达抗鸡传染性法氏囊病病毒(IBDV)重组鸡源抗体的CHO细胞株驯化为在无血清培养基中悬浮生长、高产的工程细胞株,本研究利用流式细胞仪筛选得到高产贴壁细胞株;以快速降低血清法进行驯化,使其适应悬浮培养,经过摇瓶无血清培养扩增,持续监测葡萄糖消耗情况,最终检测抗IBDV抗体的表达量。结果显示,悬浮驯化培养的CHO细胞能够在无血清培养基中稳定生长,生产周期短,接种密度为4.8×10~5cells/m L,无血清单细胞悬浮培养120 h后最大活细胞密度可达6.0×10~6 cells/m L,相同体积下抗体表达量较贴壁生长的高3倍,平均每个细胞每24 h表达量达到15 pg~20 pg。以上结果表明,贴壁生长的CHO细胞经过悬浮适应,不仅可以在无血清培养条件下快速生长,而且细胞对葡萄糖的利用率高,抗体表达量高。本研究筛选获得的细胞株节省了工艺生产中原料成本,操作方便,减少污染,易于放大,为工业化大批量生产奠定了基础。     

NDV、IBDV二联灭活苗对蛋雏鸡免疫程序研究

为探讨鸡新城疫病毒(NDV)、传染性法氏囊病毒(IBDV)二联灭活疫苗在蛋雏鸡养殖中的实际应用效果进行本试验。用鸡新城疫、传染性法氏囊二联灭活疫苗及传染性法氏囊活疫苗以不同免疫程序免疫7~14日龄蛋雏鸡,定期监测NDV、IBDV抗体水平,并在雏鸡60日龄时进行攻毒保护试验。结果表明,各二联苗免疫组NDV抗体水平在免疫后14 d即达到NDV血清学最低有效保护价标准,并维持至雏鸡60日龄。免疫后28 d,灭活苗一次性免疫各组IBDV中和抗体水平显著高于活苗两次免疫组,琼扩抗体水平与活苗两次免疫组无明显差异。雏鸡60日龄时攻毒,各免疫组均100%保护,对照组无保护。以上结果说明该二联灭活疫苗可替代IBDV活毒疫苗用于7~14日龄商品雏鸡免疫,免疫期至少为60d。     

鸡传染性法氏囊病病毒vp2基因在杆状病毒表达系统中的表达及免疫原性研究

将鸡传染性法氏囊病病毒超强毒Gx株vp2基因克隆到载体pFastBac HTA中,构建重组转座载体pFVP2,然后将其转化DH10Bac感受态大肠杆菌,将vp2基因整合到Bacmid穿梭载体中,获得重组穿梭载体BacmidVP2;通过脂质体转染将其转染Sf9昆虫细胞,获得重组杆状病毒rBacVP2。用Western blot和间接免疫荧光试验分析表明IBDV VP2蛋白在Sf9昆虫细胞获得正确表达,所表达的重组VP2蛋白分子量约50 Ku。以rBacVP2感染Sf9细胞裂解物免疫3周龄SPF鸡,在免疫后7 d可检测到ELISA抗体;免疫后14 d可检测到琼脂免疫扩散抗体。攻毒试验表明,初次免疫后14 d对IBDV超强毒株的攻击保护率为75%,2次免疫后14 d对其的攻击保护率为100%。     

Protection against infectious bursal disease virulent challenge conferred by a recombinant avian adeno-associated virus vaccine

The development and use of recombinant vaccine vectors for the expression of poultry pathogens proteins is an active research field. The adeno-associated virus (AAV) is a replication-defective virus member of the family Parvoviridae that has been successfully used for gene delivery in humans and other species. In this experiment, an avian adeno-associated virus (AAAV) expressing the infectious bursal disease virus (IBDV) VP2 protein (rAAAV-VP2) was evaluated for protection against IBDV-virulent challenge. Specific pathogen free (SPF) birds were inoculated with rAAAV-VP2 or with a commercial intermediate IBDV vaccine and then challenged with the Edgar strain. IBDV-specific antibody levels were observed in all vaccinated groups; titers were higher for the commercial vaccine group. The live, commercial vaccine induced adequate protection against morbidity and mortality; nevertheless, initial lymphoid depletion and follicular atrophy related to active viral replication was observed as early as day 14 and persisted up to day 28, when birds were challenged. No bursal tissue damage due to rAAAV-VP2 vaccination was observed. Eight-out-of-ten rAAAV-VP2-vaccinated birds survived the challenge and showed no clinical signs. The bursa:body weight ratio and bursa lesion scores in the rAAAV-VP2 group indicated protection against challenge. Therefore, transgenic expression of the VP2 protein after rAAAV-VP2 vaccination induced protective immunity against IBDV challenge in 80% of the birds, without compromising the bursa of Fabricius. The use of rAAAV virions for gene delivery represents a novel approach to poultry vaccination.     

鸡白细胞介素-6的原核表达及其对疫苗免疫增强效果的研究

本研究采用PCR技术,扩增、克隆了鸡白细胞介素-6（ChIL-6）成熟肽基因并进行原核表达,对表达的鸡白细胞介素-6组蛋白（rChIL-6）通过尿素变性、复性液复性、PBS溶液透析等步骤进行纯化,经测定rChIL-6的促小鼠脾脏淋巴母细胞的增殖能力来评估重组蛋白的生物学活性;用4个不同浓度的rChIL-6在不同部位与NDV活疫苗、AIVH5N1灭活疫苗同时肌肉注射来评价其免疫增强作用。研究结果表明,表达rChIL-6为25ku左右,经纯化后的蛋白纯度在95%以上;100pg/mLrChIL-6注射组具有较高的促小鼠脾淋巴母细胞增殖活性;不同浓度的rChIL-6注射组对NDV和AIVH5N1疫苗都具有较好的免疫增强作用,但对其免疫抗体产生时间及抗体滴度峰值出现和维持时间均没有明显的影响,其中0.03μg/mLrChIL-6浓度注射组的对鸡疫苗的免疫增强作用最明显,过高或过低浓度的rChIL-6对疫苗免疫增强作用不显著。这些研究为新型免疫增强佐剂开发以及鸡免疫失败性疫病的预防和治疗奠定了基础。     

安徽省鸡传染性法氏囊病流行病学初步调查

应用RT-PCR诊断方法,对2003年9月~2005年3月间来自安徽省不同地方的52个鸡群,共154羽病/死鸡的法氏囊样品进行了检测。结果表明,传染性法氏囊病(IBD)在安徽各地普遍流行,所有检测病例的平均阳性率为40.91%。不同的日龄段平均阳性率差异极显著(P<0.01),其中30日龄以下阳性率最高(35/75),60日龄以上阳性率最低(0/16)。不同品种的鸡均可检测出IBDV,品种间阳性率差异显著(P<0.05),其中江淮麻鸡阳性率最高(25/45),土鸡阳性率最低(3/15)。不同地区的病例均检测出IBDV,但阳性率差异不显著(P>0.05),其中合肥地区阳性率最高(23/47),宣城地区阳性率最低(5/18)。     

鸡贫血病—法氏囊病疫苗免疫母鸡后子代雏鸡的免疫学变化

本项目应用现代免疫学新技术对鸡传染性贫血病（CIA）-传染性法氏囊病（IBD）疫苗联合免疫母鸡后,其子代雏鸡外周血液T、B细胞数量和IgG、IgM、IgA含量法及法氏囊、胸腺、脾脏、盲肠扁桃体、哈德尔腺的T细胞和IgG、IgM、IgA抗体生成细胞数量以及泪液、气管液、胆汁、肠液的IgA、IgM、IgG含量的变化进行了动态研究。结果发现,CIA-IBD疫苗联合免疫母鸡后,其子代雏鸡外周血液、免疫器官组织和局部体液的上述各项指标均不同程度地高于未免疫的相应对照雏鸡。表明CIA-IBD疫苗免疫母鸡后,其子代雏鸡的体液免疫和细胞免疫功能明显增强,而CIAV-IBDV强毒攻击后,未免疫的子代雏鸡,其外周血液,免疫器官组织和局部体液的各项免疫学指标均明显低于疫苗免疫攻毒的子代雏鸡,这与未免疫雏鸡缺乏特异性抗体,强毒攻击后,雏鸡免疫器官组织广泛损害,淋巴细胞变性坏死等有关。