新城疫单抗酶联免疫试剂盒的完善及其应用

对初步研制的新城疫 (ND)单抗酶联免疫 (EL ISA)试剂盒进行了完善。以 2 .5 m g/ L 的抗 NDV腹水单抗 M2 2 包被酶标板 ,4 % FCS- PBS溶液为阴性对照 ,聚乙二醇 (PEG 10 0 0 0 )浓缩 L a Sota病毒液为阳性对照 ,P/ N≥ 2 .5为临诊样品阳性判定标准 ,对纯化的新城疫病毒 (NDV)蛋白的检出限为 2 μg/ L。用该试剂盒跟踪监测免疫鸡群中个体感染ND强毒后排毒动态 ,并平行测定血凝抑制 (HI)抗体。跟踪采集 2免疫鸡群泄殖腔棉拭和血液一一对应样品 2 317份 ,2实验鸡群棉拭样品 36 30份 ,血液样品 14 4 0份。结果表明 ,HI效价在 2～ 14之间的个体均可检出强毒 ,但 HI在 6以下的个体检出率最高 ,而排毒个体的高水平抗体是强毒感染刺激所致。个体感染强毒后一般排毒 1～ 3周 ,其中 HI抗体水平低者排毒时间相对较长 ;个体在感染后 3～ 6 d和 11～ 14 d出现 2个排毒高峰。强毒一旦侵入鸡群便可在群内巡回传播 ,长期维持下来。 HI抗体在感染初期先下降 ,然后迅速上升 ,且感染前低者上升速度较快 ,幅度亦较大     

免疫鸡群感染新城疫强毒后排毒及其HI抗体消长

给两组二次新城疫(ND)疫苗免疫的鸡群分别于40日龄、60日龄经胸肌注射强毒株F48E8(剂量为0．25mL),观察排毒和其HI抗体效价。结果表明：鸡在感染强毒后第2天开始排毒,在第3～6天和第11～14天出现两次排毒高峰,整个排毒过程可达3周,其中抗体水平较低者排毒时间相对较长,频率较高;强毒感染初期,个体HI抗体先下降1～3个滴度,然后上升,且感染前低者上升速度较快,幅度亦较大。     

免疫鸡群中新城疫强毒感染流行趋势

新城疫 ( ND)仍是目前我国分布最广、危害最严重的禽病之一 [1] 。 ND的临诊表现差异很大 ,从不明显感染到高度致死不等 ,其严重程度主要取决于感染病毒的毒力和鸡群的免疫状态[2 ] 。虽然国内几乎所有商业鸡群均需多次使用 ND疫苗 ,但免疫鸡群仍屡屡发生 ND。因此 ,控制和减少免疫鸡群中 ND强毒感染与传播成为我国鸡病防制工作中亟待解决的问题 [3 ]。本试验运用 ND单抗酶联免疫试剂盒作为 ND流行病学调查手段 ,对华东地区部分免疫鸡群中 ND强毒感染进行监测 ,结合测定个体 HI抗体 ,探讨免疫鸡群中 ND强毒感染流行原因和趋势 ,为制…     

免疫鸡群新城疫强毒感染及其与HI抗体的相关性研究

为调查新城疫强毒(vNDV)在免疫鸡群中的感染情况及其与抗ND抗体效价的相关性,用自行建立的检测vNDV的荧光定量RT-PCR(RRT-PCR)对采自川、渝两地36个免疫鸡群的360份肛门棉拭子进行检测,同时采血测定其血凝抑制抗体效价.结果共检出5个vNDV 阳性鸡群,每个阳性鸡群检出1例阳性样本,经病毒分离鉴定证实为NDV;36个受检鸡群HI抗体平均效价在4.5～10.1,标准偏差(SD)在0.70～3.19,变异系数在9.5%～44.3%.当SD大于2.0时,vNDV感染鸡群阳性率为50%,6(5/10),当CV大于32%6时,vNDV感染鸡群阳性率为62.5%(5/8);vNDV感染个体HI抗体效价分别为210、29,28,26,23;阳性样本RT-PCR产物经测序分析证实为vNDV F基因序列,发育进化分析显示,2株vNDV属基因Ⅶ型,3株属基因Ⅸ型.研究结果表明,RRT-PCR适合vNDV感染及传播的监测和分子流行病学调查;免疫鸡群是否感染vNDV可能与个体HI抗体水平高低无关,而与群体HI抗体离散度有关,SD和CV值有助于判定鸡群感染vNDV的风险.     

新城疫病毒HI抗体效价与流行株感染排毒之间的相关性

为了分析免疫鸡群中新城疫强毒感染流行的原因,明确抗体效价与流行株感染排毒率之间的关系,本研究以LaSota为抗原制备新城疫灭活疫苗,并以0.02mL和0.4mL的量分别免疫3周龄的SPF鸡10只。免疫后7、14、21d分别测定免疫鸡血清中的抗体HI效价。免疫后21d以基因Ⅶd亚型新城疫流行株JS5/05进行攻毒,攻毒后每天观察试验鸡的临床症状,并于攻毒后3、5、7d采集试验鸡的喉气管与泄殖腔棉拭样品进行病毒分离,结果显示,免疫3周后0.02mL和0.4mL疫苗免疫组鸡的血清HI抗体平均效价分别为5.4log2和8.2log2;0.02mL免疫组在攻毒后的排毒率达到100%,且排毒时间较长,而0.4mL免疫组在攻毒后的排毒率明显降低,且排毒时间较短。上述结果表明新城疫抗体效价与流行株感染排毒率之间存在明显的负相关。     

非典型鸡新城疫的病因研究初报（一）

应用血清学、生物学方法、从我省产蛋量下降、HI抗体在1：128以上的疑似非典型鸡新城疫（CND）病鸡群中分离到1株病毒，经鉴定和致病力测定，明确是1株ND强毒，毒力与F48E9相近，然后采用该株野毒感染不同抗体水平的3批蛋鸡及肉鸡，结果3批蛋鸡均是低抗体滴度组（1：10－1：40）发生了CND症状，感染后6－7天开始产蛋停止，经15－17天后才恢复产蛋，高抗体滴定组（1：80以上）无明显变化，无抗体对照组出现典型鸡新城疫变化，全部死亡；病毒感染后第6、10天监测HI抗体，结果低滴度组第6天达1：80－1：160，第10天达1：160－1：1280。试验结果表明，我们分离到的NDV，既可引起典型的ND，也可引起非典型的ND；高HI抗体蛋鸡产生非典型鸡新城疫的病因之一，是由于存在低抗体鸡群而发生了CND，其高HI抗体有竞争是由于强毒感染后产生的抗体。     

鸡新城疫免疫鸡群强毒感染与HI抗体水平关系的研究

应用RT-PCR方法对鸡新城疫4种不同免疫程序鸡群NDV强毒的感染进行监测。结果显示,鸡新城疫弱毒疫苗和油乳剂灭活疫苗同时接种的免疫鸡群NDV强毒的感染率最低。同时应用HA-HI试验对这4群鸡进行平行抽样检测其抗体效价,发现鸡群免疫水平整齐且平均HI效价在11 log2以上时,免疫鸡群基本不感染鸡NDV强毒。     

麻鸭感染新城疫病毒HI抗体效价的比较研究

以 NDV 强毒,弱毒分别感染麻鸭,30天后检测其血清 HI 抗体水平、并与自然感染 NDV 动物、对照组动物血清 HI 抗体水平作比较;强毒感染动物血清与其所产蛋卵黄上清 HI 抗体水平作比较.结果表明.麻鸭群为 NDV 野毒感染;鸭群血清 HI 抗体水平检涮,以50%以上检样出现7(log_2)以上的 HI 效价,可作为鸭群已受 NDV 野毒感染的标志;鸭卵黄不宜替代鸭血清作 NDV HI 抗体检测.     

中药预防雏鸡新城疫的试验观察

在雏鸡日粮中添加1％的中药复方添加剂，于7日龄时用NDⅡ系疫苗滴鼻；于15日龄、30日龄、60日龄时，分别测血清HI抗体效价，30日龄时让供试鸡自从感染ND强毒．其结果，添加中药复方添加剂组雏鸡血清HI抗体效价高于对照组，但差异不显著（P＞0．05）;自然感染ND强毒后，添加中药复方添加剂组雏鸡成活率明显高于对照组，统计分析有显著差异性（P＜0．05）。     

新流二联浓缩疫苗的应用效果

通过使用新城疫和禽流感的二联浓缩疫苗免疫生产鸡群，分别在免疫后的不同时间进行翅静脉采血，检测鸡群中ND、AI（H9）HI的血清抗体水平。结果显示，免疫组鸡的抗体效价3周达到高峰，ND达到8log2以上，AIH9达到9log2以上，维持在较高水平达20周以上。结果表明浓缩疫苗在肉种鸡上的应用效果较好。     

免疫鸡群感染新城疫强毒后的排毒规律

将经过２次新城疫（ＮＤ）基础免疫的ＳＰＦ鸡,随机分成５组,每组８只,在６０日龄时分别用Ｆ４８Ｅ８、Ｔｅｘａｓ、Ｈｅｒｔ３３ＮＤ强毒和Ｌａｓｏｔａ弱毒经口腔接种０．２５ｍＬ／只,１２ｈ后采取泄殖腔棉拭,利用ＮＤ强毒快速诊断试剂盒进行排毒的动态监测,并在攻毒前期、中期、后期测定各试验组ＳＰＦ鸡的ＨＩ效价。试验第３天开始排毒,２０ｄ结束,其间在６～７ｄ和１１～１３ｄ出现２次排毒高峰。ＨＩ抗体在初期先是降低１个滴度,后期开始升高而且离散度不断增大。     

SIMULATED NATURAL INFECTION OF CHICKENS WITH AUSTRALIAN LENTOGENIC NEWCASTLE DISEASE VIRUS AND SUBSEQUENT CHALLENGE WITH VIRULENT VIRUS

A total of 291 eight-week-old chickens were exposed to chickens infected with either of two Australian lentogenic strains (V4 and AVL NDV-1) of Newcastle disease virus (NDV). At 3 weeks after exposure, all chickens exposed to V4 infected chickens had developed haemagglutination-inhibition (HI) antibody. All chickens exposed to AVL NDV-1 virus infected chickens had developed HI antibody 5 weeks later. This sudden late appearance of HI antibody, to titres higher than those observed with V4 chickens, was explained by V4 virus being introduced to the AVL NDV-1 group of chickens. When groups of these chickens were challenged with Roakin virus (mesogenic NDV) at 3 weeks and Fontana 1083 virus (viscerotropic velogenic NDV) and Texas GB virus (neutrotropic NDV) at 3, 5, 10 and 21 weeks only three chickens developed clinical illness one of which died. These chickens were one AVL NDV-1 chicken contact challenged with Fontana 1083 virus at 3 weeks, one V4 chicken oronasally challenged with Texas GB virus at 5 weeks and one V4 chicken challenged oronasally with Fontana 1083 virus at 10 weeks. Susceptible non-vaccinated chickens died soon after challenge. Challenge by oronasal infection with 10(7.0) ELD50 of virus or contact with susceptible infected chickens enabled virulent virus to be isolated from most chickens and was accompanied by a large anamnestic increase in serum HI antibody.     

AEROSOL VACCINATION OF CHICKENS WITH THE V4 STRAIN OF NEWCASTLE DISEASE VIRUS

SUMMARY Two-week-old chickens, free of detectable maternal antibody to Newcastle disease virus (NDV), or with low levels of maternal antibody, were vaccinated with the V4 strain of NDV. Haemagglutination inhibition (HI) antibodies were determined at intervals after vaccination. Two hundred chickens were vaccinated by exposure to an aerosol, a dose of 10⁶ 50% embryo infectious doses (EID⁵⁰) being allowed per chicken. Forty unvaccinated chickens were placed in direct contact with vaccinated chickens. Most of the vaccinated chickens and the incontact chickens had developed HI antibodies of titre ≥ 8 within 2 weeks of vaccination. The HI antibodies in many chickens persisted for at least 8 weeks. Control chickens in a shed 15 metres from the shed containing the vaccinated chickens did not develop HI antibodies to NDV. NDV could be isolated from some vaccinated chickens for 15 days after vaccination. An aerosol dose of 10⁵EID₅₀ per chicken failed to induce a serological response in 2 groups of 40 chickens each. HI antibodies were produced in 1 of 2 groups, each of 40 chickens, vaccinated with 10⁶EID₅₀ and in both of 2 groups of 40 chickens each vaccinated with 10⁷EID₅₀. Duplicate groups of 40 chickens were vaccinated with 10⁶EID₅₀ of V4 virus per chicken administered either as an aerosol, a coarse spray or a droplet placed in the conjunctival sac. HI antibodies were produced in all the groups of chickens.     

新城疫LaSota活疫苗对基因Ⅶ型分离株的免疫保护性试验

从山东省发病鸡群分离鉴定了一株新城疫病毒(NDV),命名为SDLY01。经蚀斑纯化后进行毒力测定和序列分析表明分离株SDLY01属于基因Ⅶ型NDV强毒。20只7日龄SPF鸡免疫新城疫活疫苗LaSot a后14 d分别用NDV标准强毒F48E8和分离株SDLY01攻毒,同时设同日龄SPF鸡为对照组,未免疫任何疫苗。攻毒后观察10 d,免疫组在攻毒后食欲、精神均正常;对照组在攻毒后2～4d发病死亡,并表现ND典型的临床症状和病理变化。攻毒后第3、5、7、9 d对免疫组试验鸡取喉头、泄殖腔棉拭进行病毒分离,F48E8攻毒组病毒分离均为NDV阴性,SDLYO1攻毒组第5 d病毒分离NDV阳性,第3、7和9d病毒分离阴性。本研究结果表明LaSot a活疫苗对F48E8和SDLY01均能提供100%免疫保护,但不能完全抑制基因Ⅶ NDV分离株在体内的复制和排毒。     

鸽新城疫病毒野毒PB9601株的致病性研究

用不同方法测试了从新城疫患鸽分离到的新城疫病毒（ＮＤＶ）野毒ＰＢ９６０１株的致病性。结果,该毒株对１日龄ＳＰＦ雏鸡的脑内接种致病指数为２．００,对６周龄ＳＰＦ鸡静脉接种致病指数为０．００;该毒株对６周龄左右鸽有很强的致病性,有的血清中已有一定量的抗鸽ＮＤＶ抗体;经ＳＰＦ鸡胚传１３代的尿囊液病毒对鸽的半数致死量约为１２个ＴＣＩＤ５０。由此认为,ＰＢ９６０１株ＮＤＶ是对鸡致病性很弱但对鸽呈高度致病性的毒株,可作为国内鸽ＮＤＶ强毒的参考株。     

CAV与REV共感染SPF鸡对疫苗免疫反应的抑制作用

用1日龄SPF鸡人工感染鸡贫血病毒(CAV)和禽网状内皮增生病病毒(REV),探讨病毒感染对鸡体疫苗免疫反应的影响。结果表明,在用禽流感病毒(AIV,H5和H9)疫苗免疫后,CAV与REV单独感染均显著抑制了鸡体对H5和H9亚型禽流感病毒灭活疫苗的HI抗体反应,在CAV与REV共感染后,这种抑制作用更为明显。CAV单独感染后鸡体对新城疫病毒(NDV)和传染性法氏囊病病毒(IBDV)疫苗的免疫反应受到抑制,但与对照组在统计学上的差异不显著,然而,CAV可以显著加重REV感染对鸡体在NDV和IBDV疫苗免疫后抗体反应的抑制作用。从而证实CAV与REV共感染在疫苗免疫抑制上有协同作用。     

Generation of a genotype VII Newcastle disease virus vaccine candidate with high yield in embryonated chicken eggs

To generate a genotype VII Newcastle disease virus (NDV) vaccine with high yield in embryonated chicken eggs, we selected genotype VII NDV strain JS5/05, which possesses a high virus titer in embryos as the parental virus. Using reverse genetics, we generated a genetically tagged derivative (NDV/AI4) of JS5/05 by changing the amino acid sequence of the cleavage site of the F0 protein. Pathogenicity tests showed that NDV/AI4 was completely avirulent. NDV/AI4 was genetically stable and replicated efficiently during 10 consecutive passages in embryos. More importantly, serologic assays showed that oil-emulsion NDV/AI4 induced higher hemagglutination inhibition (HI) titers against the prevalent virus than oil-emulsion LaSota vaccine in chickens and geese. Moreover, NDV/AI4-induced HI titers rose faster than those elicited by LaSota in chickens. Both NDV/AI4 and LaSota provided protection against clinical disease and mortality after the challenge with the genotype VII NDV strain JS3/05. However, NDV/AI4 significantly reduced virus shedding from the vaccinated birds compared to LaSota. Taken together, these results suggest that NDV/AI4 can provide better protection than LaSota and is a promising vaccine candidate against genotype VII NDV.