传染性法氏囊病沉淀抗体水平与免疫保护关系的研究

以传染性法氏囊病(IBD)基因工程重组亚单位油乳剂疫苗及其重组活载体疫苗实验室试制品免疫SPF鸡,以法氏囊眼观病变及法氏囊中IBDV抗原检测结果来评定免疫保护效力。结果血清IBDV沉淀抗体水平达到1/8的鸡能抵抗IBDV强毒的攻击,低于1/4的部分鸡不能抵抗IBDV强毒的攻击。经t检验,在以血清IBDV沉淀抗体水平≥1/8所得百分率、血清IBDV沉淀抗体水平≥1/4所得百分率、法氏囊眼观变化正常鸡只百分率及法氏囊中IBDV抗原检测的阴性率之间差异都不显著。表明以血清IBDV沉淀抗体水平≥1/4所得百分率与法氏囊中IBDV抗原检测的阴性率和法氏囊眼观变化正常鸡只百分率一样,适用于评定攻毒免疫保护效力。     

试纸探针条检测鸡传染性法氏囊病疫苗中病毒含量的试验

试纸探针条可识别市售的国产或进口鸡传染性法氏囊病（IBD）代表性疫苗毒株及其鸡胚培养毒或细胞培养或其接种鸡的囊毒，并可检出经甲醛灭活的IBD标准强毒、田野囊毒、鸡胚培养毒或细胞培养毒。活的或灭活待检疫苗水溶液中IBDV的含量越高，试纸探针条上阳性线显现越快、颜色越深且明显；在30分钟的结果观察时间内，该条可检出每0.1mL样品中800ELD50或10^7.0TCID50的病毒含量，该条检测灭活IBDV的效价是AGP测定效价的32倍以上，试纸探针条为估价疫苗中IBDV的含量或疫苗质量提供了一条简便、快速、经济的新途径。     

传染性法氏囊病免疫保护试验中3种评定指标的比较

100只SPF鸡均分为5组，A、B、c3组免疫后攻毒，接种3批次自制的IBD基因工程重组亚单位油乳剂疫苗；D组不免疫不攻毒，E组不免疫而攻毒。免疫后第22天，感染IBDV强毒株BC-6／85。攻毒后第4天，将所有存活的鸡只以颈脱臼致死，收集法氏囊，以3种方法和指标（法氏囊眼观病变；法氏囊显微病变；法氏囊中IBDV抗原检测）进行分析，以评定免疫保护率。结果显示，以这3种方法和指标评定，A组免疫保护率为90％，B、C组免疫保护率均为95％；试验鸡A3、A14、B7、C16、E1～E20，法氏囊眼观病变明显，法氏囊显微病理损伤评分为3～5分，琼脂免疫扩散试验检测法氏囊中IBDV抗原均为阳性；其他试验鸡，法氏囊眼观无明显异常，法氏囊显微病理损伤评分在3分以下，琼脂免疫扩散试验检测法氏囊中IBDV抗原均为阴性。结果表明，这3种方法和指标在IBD疫苗免疫保护试验评定中具有很好的一致性。     

鸡传染性法氏囊病毒超强毒株的分离及生物学特性鉴定

用SPF鸡及鸡胚，从吉林某鸡场病死鸡的法氏囊组织到一株超强毒株（J20株）。此病毒不能凝集鸡的红细胞，可以被IBDV标准阳性血清检出IBDV抗原。通过对J2001分离毒株进行回归实验，测定病毒效价EID50达10^6/0.2ml，发病率为100％，死亡达70％；病毒理化特性试验，电镜观察等证实J20株为鸡传染性法氏囊病超强毒株。     

中药制剂"瘟毒杀"对IBDV、NDV和AIV的抑制试验

选10日龄SPF鸡胚,进行了瘟毒杀煎液对鸡胚人工感染NDV、IBDV和AIV抑制试验.结果显示,瘟毒杀药液(1g/ml)2倍、4倍和6倍稀释,对感染NDV强毒的鸡胚保护率达到100%、75%和62.5%,比NDV强毒对照组分别提高87.5、62.5和50个百分点;对感染AIV强毒的鸡胚保护率达到87.5%、62.5%和50%,比AIV强毒对照组分别提高75、50和37.5个百分点;对感染IBDV强毒的鸡胚保护率达到100%、87.5%和62.5%.比IBDV强毒对照组分别提高75、62.5和37.5个百分点.NDV强毒接种组鸡胚尿囊液NDV血凝效价极显著高于NDV强毒+瘟毒杀药液组尿囊液NDV血凝效价;AIV强毒接种组鸡胚尿囊液AIV血凝效价极显著高于AIV强毒+瘟毒杀药液组尿囊液AIV血凝效价.结果表明,瘟毒杀对NDV、AIV和IBDV有较强的抑制作用.     

异源抗原在建立间接酶联免疫试验(ELISA)检测传染性法氏囊病毒抗体中的应用

本文利用vero细胞增殖的IBDN抗原建立了间接酶联免疫吸附试验（ELISA），用于定量检测鸡传染性法氏囊病毒（IBDV）抗体。该法快速，敏感性高、特异性强、重复性好。同时，通过30份血清样品ELISA致价（ET）的对数值，（logET）与血清P／N值（待检血清OD也与阴性血清OD值之比）的线性回归分析，得直线方程y=3．0589＋0．0739x（r=0．9174），从而血清样品的ET可通过血清单一稀释度的P／N值来计算。用不同抗原来源作ELISA表明，从vero细胞增殖的抗原比从鸡胚成纤维（CEF）细胞增殖的抗原可提高检测血清OD值近20％，表现出异源抗原县有更高的特异性。     

传染性法氏囊病病毒感染细胞内源性microRNA表达差异分析

细胞内源性microRNA（miRNA）在宿主与病原相互作用过程中发挥着重要的调节功能。为了分析细胞内源性miRNA与传染性法氏囊病病毒（IBDV）的相互作用,用IBDV弱毒和强毒分别感染鸡胚成纤维细胞（CEF）和SPF鸡,24h后,取感染CEF和法氏囊组织,提取细胞总RNA,用Hy3/Hy5双色荧光标记,与miRNA芯片杂交,进行芯片内标准化、芯片间标准化、表达差异比较以及聚类分析。结果显示：在IBDV弱毒感染的CEF中,有17个细胞内源性miRNA表达上调,17个miRNA表达下调;在IBDV强毒感染的鸡法氏囊组织细胞中,有30个细胞内源性miRNA表达上调,18个miRNA表达下调。根据表达差异显著的miRNA序列设计引物,用荧光定量RT-PCR方法验证芯片检测结果,2种方法的检测结果一致。结果表明,IBDV感染可诱导细胞内源性miRNA表达变化,这些上调或下调的miRNA能调控细胞内多种代谢和信号传导途径发生异常,引起细胞及组织器官发生病理学变化。     

法氏囊病毒感染对肉鸡内分泌机能的影响

用鸡传染性法氏囊病毒（ＩＢＤＶ）弱毒疫苗免疫和强毒感染艾维菌鸡，采用放射免疫分析法（ＲＩＡ）测定血浆甲状腺素（Ｔ３，Ｔ４）、促肾上腺皮质素（ＡＣＴＨ）和皮质酮（Ｆ）来研究ＩＢＤＶ感染对肉鸡内分泌机能的影响。结果表明：ＩＢＤＶ弱毒疫苗免疫使肉鸡产生高效价抗体，血浆ＡＣＴＨ含量升高；ＩＢＤＭ强毒感染导致肉鸡血浆Ｔ２、Ｆ急剧升高，法氏囊肿大，血清中也出现ＩＢＤＶ抗体，但效价显著低下；而ＩＢＤＶ强毒感染曾     

传染性法氏囊病病毒检测系统的建立

为了建立传染性法氏囊病病毒(IBDV)检测系统,本试验在同一个试验技术平台上对9种IBDV检测方法进行了比较分析.IBDV野毒株盲传4～5代可适应于鸡胚和鸡胚成纤维细胞,死亡胚体水肿、出血,病变细胞圆缩、脱落.4个血清Ⅰ型1BDV毒株,用交叉中和试验可进一步分为3个血清亚型.用RT-PCR/SSCP技术分析4个毒株,扩增产物的电泳迁移率有差异.AGP、间接ELISA、夹心抑制ELISA以及中和试验等4种方法同时检测不同来源血清样品中的IBDV抗体,其他3种方法比AGP具有更高的敏感性,敏感度相差104倍.用AGP检测法氏囊组织样品,抗原效价可达到1:10,而细胞培养物和病鸡粪便则没有出现沉淀线;3种样品用夹心ELISA、RT-PCR、cDNA探针斑点杂交、RT-PCR/SSCP检测以及病毒分离等5种方法检测,均为阳性.     

同胚接种IBDV和NDV制造二联弱毒苗

用鸡传染性腔上囊病病毒（IBDV）和新城疫病毒（NDV）接种同一鸡胚收取种毒，试制IBDV、NDV二联弱毒冻干疫苗，安全性和效力检验均达到这2种单苗《规程》规定的标准，证明2种病毒在鸡体人产生互不干扰抗体。     

Development of a one-step strip test for the diagnosis of chicken infectious bursal disease

A rapid diagnostic strip for chicken infectious bursal disease (IBD) was developed based on membrane chromatography using high-affinity monoclonal antibodies directed to chicken infectious bursal disease virus (IBDV). The diagnostic strip has high specificity for detection of chicken IBDV antigen and recognizes a variety of the virus isolates, including virulent and attenuated strains, with no cross-reactivity to other viruses, such as Newcastle disease virus, Marek's disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and egg-drop-syndrome virus. The results showed that its specificity was highly consistent with the agar-gel precipitation test (AGP). The diagnostic strip detected as low as 800 median egg lethal dose (ELD50) viruses in the IBDV BC6/85-infected sample, which was comparable with AC-ELISA (400 ELD50) and 32 times more sensitive than the AGP test (2.56 x 10(4) ELD50). In experimental infection, IBDV was detected in the bursa as early as 36 hr postinfection with the diagnostic strip before the clinical signs and gross lesions appeared. It takes only 1-2 min to do a strip test to detect chicken IBDV antigen after the specimen is grounded in a whirl pack with finger massage.     

安徽地区IBDV超强毒株的分离鉴定和VP2基因序列分析

经鸡胚绒毛尿囊膜(CAM)接种、易感鸡接种试验、电镜观察,从安徽地区疑似病鸡的法氏囊组织分离到3株传染性法氏囊病毒。分离株人工感染4周龄鸡,致死率分别为92%、83%、67%。接种9～10SPF鸡胚测得的鸡胚半数致死量(ELD50)分别为10-6.8/0.2mL、10-5.4/0.2mL、10-4.6/0.2mL。应用Nested-PCR分别对3株分离株VP2基因高变区进行克隆测序和序列分析,结果表明:3个分离株与国内外参考超强毒株的核苷酸同源性为97.2%～99.5%,氨基酸同源性为99.3%～100%,VP2高变区核苷酸和推导的氨基酸符合传染性法氏囊病病毒超强毒株特征。     

应用Mab-basedAC—ELISA法对鸡IBDV分离株的抗原特性分析

为了解传染性法氏囊病病毒（IBDV）的抗原变异分子基础,采用Mab—basedAC—ELISA方法,利用11株单抗对2株河北IBDV分离株进行了抗原特性分析,并比较了超强毒株和弱毒株的VP2高变区序列的分子特征。结果显示：强弱毒株的抗原位点结合单抗的能力明显不同,除了Mab3、Mab9外,其余9个单抗的结合能力,超强毒株要高于弱毒株。超强毒株HeB-If缺少了Mab3结合位点,弱毒株HeB-bdjz缺少了Mab3、Mab6结合住点,这说明强弱毒株不仅在序列上有差异,而且在抗原位点的缺失上也存在差异。本试验为控制IBD和疫苗改进具有重要意义。     

Comparison of the dot blot hybridization assay with antigen detection assays for the diagnosis of infectious bursal disease virus infections

The use of cDNA probes as diagnostic tools for detecting infectious bursal disease virus (IBDV) antigens was compared with the agar-gel precipitin (AGP) and immunofluorescence (IF) assays. Specific-pathogen-free chickens were inoculated with the STC or IN strains of IBDV in three separate experiments. Tissue samples were collected at various intervals postinoculation (PI) and examined for viral antigens using the IF assay, the AGP assay, and the hybridization assay. Viral antigen was detected by the AGP assay for a period of approximately 3 days beginning 2 days PI and by the IF assay for approximately 4 days beginning 2 days PI. Virus was detected by the hybridization assay through the length of all the experiments (longest experiment was 24 days) beginning 1 day PI. These results suggested that the hybridization assay is more sensitive than the IF assay and the AGP assay.     

Rocket Immunoelectrophoresis in the Diagnosis of Infectious Bursal Disease

The rocket immunoelectrophoresis (RIE) test was used for the qualitative detection and quantitative estimation of infectious bursal disease virus (IBDV) specific antigen in experimentally infected chickens and samples collected from suspected outbreaks. The IBDV specific antigen was detected in the bursae of experimentally inoculated chickens up to 5 days post infection (PI) by the agar gel precipitation (AGP) test and 7 days PI by the RIE test. The RIE detected IBDV specific antigen in a significantly greater number of samples collected from the field outbreaks than the conventional AGP test. Exudative bursae were found to have a higher antigen content than haemorrhagic bursae and are recommended as the material of choice for diagnosis of IBD. This test could also be used to quantify IBDV specific antigen in commercial killed vaccines.     

用免疫胶体金试纸膜快速检测IBDV的研究

用免疫胶体金试纸膜分别检测了GX8／99株超强毒传染性法氏囊病病毒(vvIBDV)不同传代毒、全国部分省市IBDV地方野毒、鸡胚传代毒、细胞传代毒、临床病料及其他病原材料，试验研究结果表明免疫胶体金试纸膜法与琼脂扩散试验(ACP)相比，具有特异、敏感、快速、简单等特点，不需要特殊的专业技能和其他试剂，能够识别不同的IBDV毒株，包括强毒和弱毒。为IBDV开辟了一条新的、快速的检测途径。     

An immunoperoxidase monoclonal antibody stain for rapid diagnosis of infectious bursal disease

Cell smears of chicken-embryo-fibroblast (CEF) cultures and bursa of Fabricius from chickens experimentally infected with six different strains of infectious bursal disease virus (IBDV) were examined for the presence of IBDV by the avidin-biotin-peroxidase complex method of immunoperoxidase (IP) staining using a monoclonal antibody specific for IBDV designated BK70. IBDV of different strains and serotypes were readily detected by the IP method in cell smears prepared from infected CEF cultures and from bursas. Bursal cells were positive for IP stain in most of the infected bursas (87.5%), despite their mild IBD lesions. Positive IP staining of bursal smears was well correlated with the recovery of IBDV from the bursas and with IBD lesions in the bursas. IP stain with a monoclonal antibody (BK70) appeared potentially useful for rapid and definitive diagnosis of IBD.     

鸡传染性法氏囊病快速诊断试纸条的研制及特性测定

以高亲和力和高特异性IBDV单克隆抗体为基础,成功的研制出IBDV快速诊断试纸条,试验结果表明IBD试纸与鸡的NDV、ALV、EDS-76、IBV、ILTV、MDV无交叉反应。IBD试纸条具有良好的特异性和敏感性,其敏感性与琼扩试验相比,则提高了32—64倍,能够识别不同的IBDV毒株包括强毒和弱毒。与ELISA的阳性符合率为100％。对于人工感染IBDV鸡,感染后36h在感染鸡的法氏囊即可检出IBDV,从检测到获得结果仅需要1-2min,IBD试纸具有特异、敏感、快速、简单等特点,不需要任何专业技能和其他试剂,实现了鸡病诊断的一步法。     

Comparative immunopathogenesis of mild,intermediate, and virulent strains of classic infectious bursal disease virus

Differences in the immunopathogenesis of several strains of infectious bursal disease virus (IBDV) were compared. The strains included a virulent virus (IBDV-IM) and three vaccine viruses that included an intermediate vaccine virus (IBDV-B2) and two mild vaccine viruses (IBDV-Lukert and IBDV-BVM). The most significant differences were found in the systemic effects of these strains. In comparison with other strains, IBDV-IM antigen was detectable for up to 8 days postinfection (PI) in lymphoid tissues that included spleen and cecal tonsils, whereas only a few IBDV-B2- and IBDV-Lukert- and no IBDV-BVM-inoculated birds had detectable IBDV antigen in these tissues. IBDV-IM induced systemic circulating nitrite levels in over 86% of the birds at days 2 and 3 PI. IBDV-IM suppressed most vigorously the splenic mitogenic response on days 3-8 PI. Among the three vaccine strains, IBDV-B2 was the most virulent of the three, inducing a significant suppression of the mitogenic response (P < 0.05) and the most vigorous lesions in the bursa of Fabricius with the highest possible lesion score of 4 at 3 days PI (P < 0.05). IBDV-BVM was the mildest strain, not inducing any detectable lesions in lymphoid tissue at the tested time points. Whereas all IBDV-BVM-inoculated and 67% and 33% of the IBDV-Lukert- and IBDV-B2-inoculated birds, respectively, had detectable IBDV antigen in the bursa at 4 days postchallenge, none of the IBDV-IM-inoculated birds was positive for IBDV by immunohistochemistry. IBDV-IM induced the highest enzyme-linked immunosorbent assay (ELISA) antibody levels detected at days 8-29 PI (P < 0.05) and the best protection against challenge virus replication in comparison with IBDV-B2 and IBDV-Lukert. Only one of five IBDV-BVM-inoculated birds developed anti-IBDV ELISA antibodies at 29 days PI, and none of the birds was protected against IBDV challenge. We speculate that better protection with more virulent strains was due to more systemic antigenic stimulation on the basis of higher replication of IBDV in extrabursal lymphoid tissues. Interestingly, IBDV-IM did not differ from IBDV-B2 and IBDV-Lukert in its ability to induce T cell accumulation in the bursa at 8 days PI and local interferon-gamma induction from days 2 to 5 PI. These results suggested that the local T cell events in the bursa alone may not be indicative of a rapid and protective immune response.