抗传染性法氏囊病病毒单克隆抗体的特性研究——传染性法氏囊病病毒抗原的培养与提纯

在制备传染性法氏囊病病毒 (ＩＢＤＶ)单克隆抗体前 ,首先要提纯ＩＢＤＶ抗原 ,然后才能免疫ＢＡＬＢ/ｃ小鼠 ,检测和筛选阳性孔 ,克隆阳性孔并制备单克隆抗体。本实验采用蔗糖密度梯度离心法和超滤膜超滤浓缩法提纯ＩＢＤＶ ,并以纯化抗原免疫ＢＡＬＢ/ｃ小鼠 ,成功地制备出抗ＩＢＤＶ单克隆抗体。1　材料和方法1.1　材料ＩＢＤＶＤ78、ＮＤＶ、ＩＢＶ和ＭＤＶ均由哈兽研生物技术国家重点实验室提供 ;ＳＰＦ鸡胚 ,购自黑龙江省生物制品一厂。1.2　鸡胚成纤维原代细胞制备按Ｈａｎｓｏｎ[1] 等描述方法制备鸡胚成纤维细胞。1.3　病毒繁殖将…     

鸡新城疫,减蛋综合症,传染性支气管炎三联油佐剂灭活苗的研究：I…

分别用三个滴度的ＮＤＶ、ＥＤＳ７６Ｖ、ＩＢＶ作正交配比，制成八组不同配比的三联油佐剂灭活苗，鸡免疫后每月采血分离血清，应用ＨＩ监测其抗体，结果表明，三种病毒适当配比，不产生干扰，疫苗的免疫效果与抗原量有关。本疫苗与Ｉｎｔｃｒｖｃｔ公司生产的ＮＤ－ＩＢ－ＩＢＤ三联油佐剂灭活苗作比较试验，免疫鸡所产生的ＮＤ、ＩＢ抗体无显著性差异。应用含抗原量最低配比的疫苗，对不明原因的有产蛋率下降疫病感染史的鸡群作免     

以鸡痘病毒为载体的新城疫病毒重组疫苗的免疫效力

构建了表达速发型新城疫病毒（ＮＤＶ）融合蛋白（Ｆ）和血凝素－神经氨酸酶（ＨＮ）糖蛋白的鸡痘重组病毒ＴＲＯＶＡＣ－ＮＤＶ。ＳＰＦ鸡免疫试验表明，一次注射得组病毒有使鸡产生大量血凝抑制（ＨＩ）抗体。并能维持８周以上，而且重凤苗能诱导对经肌肉和呼吸道途径攻击ＮＤＶ的免疫力。带母滞病毒（ＦＰＶ）的免疫力，但ＮＤＶ的特异性体液应签水平降低。     

IBD疫苗抗原量对鸡体液免疫应答的影响

将含不同抗原量的ＩＢＤ油乳剂灭活疫苗免疫１７日龄雏鸡,结果：ＩＢＤ０－ＮＤ二联苗和ＩＢＤ浓缩苗免疫组难的抗体水平上升较快,免疫１１天时免疫组抗体水平显著高于免疫对照组（Ｐ〈０．０１）;３个免疫组鸡的攻毒保护率基本一致,但ＩＢＤ原菌组鸡的腔上囊损伤最为严重。试验结果表明：适当增加ＩＢＤ抗原量能刺激鸡早期体液免疫应答,提高特生免疫力。     

鸡新城疫、减蛋综合症、传染性支气管炎三联油佐剂灭活苗的研究Ⅱ、不同单位病毒配比的三联苗对鸡免疫抗体产生的影响

分别用三个滴度的ＮＤＶ、ＥＤＳ_７６Ｖ、ＩＢＶ作正交配比，制成八组不同配比的三联油佐剂灭活苗，鸡免疫后每月采血分离血清，应用ＨＩ监测其抗体，结果表明，三种病毒适当配比，不产生干扰，疫苗的免疫效果与抗原量有关。本疫苗与Ｉｎｔeｒｖｃｔ公司生产的ＮＤ－ＩＢ－ＩＢＤ三联油佐剂灭活苗作比较试验，免疫鸡所产生的ＮＤ、ＩＢ抗体无显著性差异。应用含抗原量最低口比的疫苗，对不明原因的有产蛋率下降疫病感染史的鸡群作免疫接种，冬季该场蛋鸡群普遍发生产蛋率明显下降的疫病，用本苗免疫的鸡群产蛋享未受影响。     

禽流感RT-PCR诊断法的建立

根据禽流感病毒（ＡＩＶ）Ａ／Ｃｈｉｃｋｅｎ／Ｂｒｅｓｌｉｎ／１９０２（Ｈ７Ｎ７）株的血凝素（ＨＡ）基因参考序列设计合成一对Ｈ７ＨＡ特异引物,并应用ＳＤＳ－蛋白酶Ｋ法提取病毒ＲＮＡ,建立了逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）检测ＡＩＶＲＮＡ的方法。应用此法对鸡胚培养的ＡＩＶ尿囊液、ＳＰＦ感染鸡粪便进行了检测,并与琼脂扩散试验和电镜技术作了对比。特异性试验以新城疫病毒（ＮＤＶ）、传染性法氏囊病病毒（ＩＢＤＶ）、传染性支气管炎病毒（ＩＢＶ）、减蛋综合征病毒（ＥＤＳ－７６Ｖ）等的感染鸡胚尿囊液作为对照。结果表明,ＡＩＶ尿囊液ＲＴ－ＰＣＲ全部阳性,ＳＰＦ感染鸡粪便８７份,ＲＴ－ＰＣＲ检出６９份阳性,样品处理浓缩后琼脂扩散检出１１份,电镜检测了２３份样品,仅检出２份阳性。非特异性对照样品经ＲＴ－ＰＣＲ检测全部阴性。将ＡＩＶ感染鸡胚尿囊液作１０倍系列连续稀释,可检出稀释度为１０－２的病毒尿囊液。ＲＴ－ＰＣＲ最早检出时间为感染后第３天,而琼扩和电镜均是７天。该法具有高度的特异性和灵敏性,且节约时间（只需８小时左右）,可从分子水平上对ＡＩＶ进行早期快速诊断和流行病学调查     

禽流感RT—PCR诊断法的建立

根据禽流感病毒（ＡＩＶ）Ａ／Ｃｈｉｃｋｅｎ／Ｂｒｅｓｌｉｎ／１９０２（Ｈ７Ｎ７）株的血凝素（ＨＡ）基因参考序列设计合成一对Ｈ７ＨＡ特异引物,并应用ＳＤＳ－蛋白酶Ｋ法提取病毒ＲＮＡ,建立了逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）检测ＡＩＶＲＮＡ的方法,应用此法对鸡胚培养ＡＩＶ尿囊液,ＳＰＦ感染鸡粪便进行了检测,并与琼脂扩莠试验和电镜技术作了对比,特异性试验以新城疫病毒（ＮＤＶ）,传染性氏囊病病毒（ＩＢＤ     

感染鸡贫血病毒雏鸡ND免疫后血清Igs和HI抗体的动态变化

本试验对１日龄感染鸡传染性贫血病毒（ＣＩＡＶ）雏鸡接种新城疫（ＮＤ）疫苗及其强毒攻击后外周血液免疫球蛋白ＩｇＧ、ＩｇＭ、ＩｇＡ含量和ＨＩ抗体滴度的动态变化进行研究。结果发现，ＣＩＡＶ感染雏鸡ＮＤ疫苗免疫后，其外周血液ＩｇＧ、ＩｇＭ、ＩｇＡ含量和ＨＩ抗体滴度，于接种ＮＤ疫苗后７￣２８ｄ较未感染ＣＩＡＶ的免疫对照雏鸡明显减少，表明ＣＩＡＶ感染雏鸡对ＮＤ疫苗免疫的体液免疫应答功能明显降低。ＮＤ强毒攻击后     

感染CIAV雏鸡岛免疫器官对ND疫苗的细胞免疫应答

对１日龄感染ＣＩＡＶ雏鸡接种ＮＤ疫苗后，其免疫器官组织Ｔ细胞数量的动态变化研究，结果，感染ＣＩＡＶ雏鸡ＮＤ疫苗免疫后，其胸腺和脾脏以及盲肠桃体和哈德尔腺的Ｔ细胞数量，于接种ＮＤ疫苗后较未感染ＣＩＡＶ免疫对照雏鸡明显减少，表明感染雏鸡的中枢和外周免疫器官及局部免疫组织对ＮＤ疫苗的细胞免疫应答功能降低，ＮＤ强毒攻击后，感染免疫鸡的免疫保护率明显低于未感染免疫对照鸡。     

我国部分地区NDV的分子流行病学研究

新城疫（Ｎｅｗｃａｓｔｌｅ ｄｉｓｅａｓｅ,ＮＤ）是由新城疫病毒（ＮＤＶ）引起的禽类的一种以呼吸道、消化道粘膜出血为典型症状的急性传染病,其病原——ＮＤＶ是副粘病毒科、副粘病毒亚科、腮腺炎病毒属的成员,为有囊膜的负链ＲＮＡ病毒。ＮＤＶ基因组为单股不分节ＲＮＡ,编码六种病毒结构蛋白,其中融合蛋白（Ｆ）和血凝素－神经氨酸酶蛋白（ＨＮ）构成囊膜外表面的纤突,是ＮＤＶ的功能性糖蛋白,在致病和免疫应答过程中起重要作用［１］。由于ＮＤＶ只有一种血清型,对ＮＤＶ不同毒株的差异性分析只能采用毒力、蚀斑形成能力测定等功能性试验来进行,但分型要么太细,无规律可循;要么过于粗放,无流行病学意义,使得ＮＤＶ流行病学研究一直难以突破。 近年来,随着分子生物学技术的广泛应用,使得人们在分子水平上对病毒遗传变异机制的研究取得了长足进展,发现病毒的遗传变异与疾病的流行有着密不可分的联系,并形成了病毒分子流行病学这样一门崭新学科,也为ＮＤ的流行病学研究提供了一种先进技术。已有研究表明,ＮＤＶＦ基因的遗传变异与ＮＤＶ的变异和流行密切相关,是ＮＤＶ分子流行病学研究的首选基因。 本研究根据新城疫病毒（ＮＤＶ）Ｆ基因编码区１－３７４位核苷酸序列计算其遗     

Advancement in vaccination against Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to the limitation in their efficacy, current vaccination strategies against ND need improvements. This study aimed to evaluate a new-generation ND vaccine for its efficacy in providing clinical protection and reducing virus shedding after challenge. Broiler chickens were vaccinated in ovo or subcutaneously at hatch with a turkey herpesvirus-based recombinant vaccine (rHVT) expressing a key protective antigen (F glycoprotein) of Newcastle disease virus (NDV). Groups of birds were challenged at 20, 27, and 40 days of age with a genotype V viscerotropic velogenic NDV strain. Protection was 57% and 81%, 100% and 95%, and 100% and 100% after the subsequent challenges in the in ovo and subcutaneously vaccinated chickens, respectively. Humoral immune response to vaccination could be detected from 3-4 wk of age. Challenge virus shedding was lower and gradually decreased over time in the vaccinated birds compared to the unvaccinated control chickens. In spite of the phylogenetic distance between the NDV F gene inserted into the vector vaccine and the challenge virus (genotype I and V, respectively), the rHVT NDV vaccine provided good clinical protection and significantly reduced challenge virus shedding.     

Development of a virosome vaccine for Newcastle disease virus

In an effort to protect chickens against Newcastle disease (ND), a nonreplicating virosome vaccine was produced by solubilization of Newcastle disease virus (NDV) with Triton X-100 followed by detergent removal with SM2 Bio-Beads. Biochemical analysis indicated that the NDV virosomes had similar characteristics as the parent virus and contained both the fusion and hemagglutinin-neuraminidase proteins. To target the respiratory tract, specific-pathogen-free chickens were immunized intranasally and intratracheally with the NDV virosome vaccine. This vaccine was compared with a standard NDV (LaSota) live-virus vaccine for commercial poultry. Seroconversion (> or = four fold increase in hemagglutination inhibition [HI] antibody titers) was achieved in all birds vaccinated with the virosome vaccine. Upon lethal challenge with a velogenic NDV strain (Texas GB), all birds receiving either vaccination method were protected against death. Antibody levels against NDV, as determined by enzyme-linked immunosorbent assay and HI titer, were comparable with either vaccine and increased after virus challenge. These results demonstrate the potential of virosomes as an effective tool for ND vaccination.     

重组鸡白细胞介素-6/2融合蛋白对新城疫(LaSota株)活疫苗免疫增强作用研究

为探究重组鸡白细胞介素-6/2融合蛋白(rChIL-6-Linker-ChIL-2,重组融合蛋白)对新城疫病毒(NDV)活疫苗(LaSota株)的免疫增强作用,本研究将90只SPF鸡随机分为6组,分别为PBS对照组、NDV弱毒苗对照组、rChIL-6-Linker-ChIL-2免疫增强组、rChIL-6蛋白免疫增强组、rChIL-2蛋白免疫增强组及rChIL-6+rChIL-2混合蛋白免疫增强组,将鸡白细胞介素重组融合蛋白水剂与LaSota株联合接种于SPF鸡,分别采用MTS法、流式细胞术、ELISA和HA/HI法检测接种前后不同时间各组鸡外周血及脾淋巴细胞增殖活性、鸡外周血中CD3+CD4+/CD3+CD8+T淋巴细胞的百分含量、血清中Th1/Th2型细胞因子表达水平及免疫抗体水平等指标。结果显示,接种后7～21d时,与NDV弱毒苗对照组相比,同时接种重组融合蛋白组鸡淋巴细胞增殖活性、外周血CD3+CD4+/CD3+CD8+T淋巴细胞的百分含量比值及血清中重组鸡IL-4蛋白(rChIL-4)、ChIL-6、ChIL-2、重组鸡IFN-α蛋白(ChIFN-α)、ChIFN-γTh1/Th2型细胞因子表达水平明显提升;HI免疫抗体较NDV弱毒苗对照组提高了1.0～1.9个滴度,较单一rChIL-6、rChIL-2对照组分别提高了0.2～0.7、0.2～1.1个抗体滴度。综上所述,rChIL-6-Lin-ker-ChIL-2融合蛋白对NDV(LaSota株)活疫苗在鸡体内具有明显的免疫增强效果。     

新城疫病毒外膜蛋白脂质体疫苗的实验研究

本研究以冷冻干燥法制得新城疫病毒（ＮＤＶ）外膜蛋白脂质体疫苗，两次免疫后ＳＰＦ鸡的血清ＨＩ抗体效价结果显示：ＮＤＶ外膜蛋白脂质体疫苗免疫组的算术滴度要高于仅以ＮＤＶ外膜蛋白免疫组，二者间具有是生差异，且前者以ＮＤＶ强毒株攻击后的保护率也１００％，也优于单纯以ＮＤＶ外膜蛋白免疫组。     

Effect of the Immunity Efficacy of IBD Immune Complex Vaccine on ND La Sota Vaccine

1-day-old SPF chickens and commercial Jingfen chickens were vaccinated with IBD immune complex(IC) vaccine, NDV La Sota vaccine were inoculated simultaneously every one week and every two weeks.NDV La Sota immunization alone was as the control group.At the 2nd, 3rd, 4th and 5th week post inoculation, blood samples were taken and the ND HI antibody were tested.Experimental chickens were challenged with high virulent NDV at the 5th week post inoculation, the protective rate of each group was calculated.The results showed the ND HI antibody were not significant different in the combined immunization of IBD IC vaccine priming and NDV La Sota vaccine boost and NDV La Sota vaccine alone immunization (P>0.05).The results indicated that IBD IC vaccine has no immunosuppression on NDV La Sota vaccine in SPF chickens and commercial Jingfen chickens.     

Formulation of novel nano-encapsulated Newcastle disease vaccine tablets for vaccination of village chickens

Formulation of nano-encapsulated vaccine tablet is a novel technique for the delivery of Newcastle disease (ND) vaccine to village chickens. Vaccine tablets were prepared using gelatin, trehalose and casein as thermostabilisers and binders, respectively, and each vaccine tablet contained a nominal oral dose of Newcastle disease virus (NDV) strain I-2 for a single chicken. These ND vaccine tablets maintained a titre of 10^8.5 EID₅₀/0.1 mL for 90 days at ambient room temperatures (25–34°C). When these vaccine tablets were given to village chickens, a single oral administration of the vaccine produced protective antibody response (≥3.0 log₂) against challenge with virulent NDV. The findings from the present study showed that, if the vaccine tablet formulation technique is optimised, it will allow the delivery of the ND vaccine without depending on cold chains to rural areas in tropical countries.     

Immune response to oil-emulsion vaccines with single or mixed antigens of Newcastle disease, avian influenza, and infectious bronchitis

Inactivated Newcastle disease virus (NDV), avian influenza virus (AIV), and infectious bronchitis virus (IBV) antigens were evaluated for immunological efficacy in monovalent and polyvalent vaccines. Vaccinated broilers were bled for hemagglutination-inhibition (HI) tests at 1- or 2-week intervals. Half of the chickens were challenged with the Largo isolate of velogenic viscerotropic (VV) NDV at 8 weeks post-vaccination, and the remainder were challenged with the Massachusetts 41 strain IBV at 9 weeks post-vaccination. Newcastle disease HI titers were reduced significantly (P less than 0.05) from those of monovalent control vaccine groups when IBV antigen was emulsified in mixtures with low (1-3x) concentrated NDV or NDV and AIV antigens. Avian influenza HI titers were significantly (P less than 0.05) lower than those of the control monovalent groups when highly concentrated NDV was part of the polyvalent vaccine. Infectious bronchitis HI titers were higher than those of control monovalent groups in 13 of 15 vaccine groups when IBV antigen was in polyvalent formulations. VV NDV challenge killed all non-NDV vaccinates and induced increased HI titers in NDV vaccinates but no morbidity or mortality. Sixty of 80 IBV vaccinates experienced a fourfold or greater HI titer increase following challenge. All non-IBV vaccinates seroconverted at 1 week post-challenge.     

Determination of minimum hemagglutinin units in an inactivated Newcastle disease virus vaccine for clinical protection of chickens from exotic Newcastle disease virus challenge

The potency of inactivated Newcastle disease virus (NDV) vaccines in the United States is currently determined using vaccination and challenge of experimental animals against a velogenic strain of NDV. Because velogenic strains of NDV are now classified as select agents in the United States, all vaccine potency testing must be performed in live animals under biosafety level 3 agriculture conditions. If the minimum amount of inactivated viral antigen required for clinical protection can be determined using other methods, vaccines meeting these criteria might be considered of adequate potency. The linearity of correlation between the hemagglutination (HA) assay measurement and the 50% embryo infectious dose titer ofNDV Hitchner B1 vaccine virus was determined. Correlation between hemagglutinin units (HAU) per vaccine dose, clinical protection, and antibody response was then determined using a vaccinate-and-challenge model similar to Chapter 9 of the U.S. code of federal regulations approved method for vaccine potency testing. The dose providing 50% protection of an in-house water-in-oil emulsion vaccine formulated with inactivated NDV B1 was determined to be between 400 and 600 HAU from two separate trials. A positive correlation (R2 = 0.97) was observed between antibody response and HAU per vaccine dose. Serum antibody responses from vaccinated birds indicate HA inhibition titers >2(5) log2 would provide 100% protection from morbidity and mortality and require a minimum protective dose of 1000 HAU per bird. These are the first studies to examine establishing both a minimum protective HAU content for inactivated ND vaccines and a minimum serologic response necessary to ensure potency.     

脂质体包被鸡IL-18可增强新城疫活疫苗的免疫效果

首次将重组鸡白细胞介素18(mChIL-18)用脂质体包被,将不同浓度的mChIL-18脂质体联合新城疫疫苗对鸡群进行免疫,观察其对新城疫疫苗的免疫增强作用。1日龄SPF鸡分为6组,分别为空白对照组、单纯疫苗组、新城疫疫苗+0.2mL mChIL-18组、新城疫疫苗+0.1mL mChIL-18组、新城疫疫苗+0.3mL mChIL-18组和新城疫疫苗+空载体对照组。所有免疫组鸡于7日龄免疫,免疫后每周抽取外周血及外周抗凝血,应用血凝抑制试验和流式细胞仪来检测免疫鸡的HI抗体水平及CD4+和CD8+T淋巴细胞的变化情况。并于最后一次采血后,对各组试验鸡应用F48E9标准强毒进行攻击(100ELD50.只-1)。结果表明mChIL-18脂质体的最佳剂量为0.2mL.只-1,其不仅能够显著增强机体的细胞免疫和体液免疫,而且还可以明显提高新城疫疫苗的保护率。     

从自然发病鸡中分离出一株新城疫病毒

应用电镜技术、血清学检查、生物学试验等方法,从未注射过疫苗的某自然发病鸡群分离出一株新城疫病毒。该分离株对１０日龄鸡胚的平均致死时间为１１６小时,静脉接种致病指数为０．０６,其毒力介于新城疫病毒的ＬａＳｏｔａ株与Ｖ４株之间。免疫原性试验结果表明,该分离株具有免疫后无不良反应、免疫后６天开始产生抗体、产生的抗体效价高、对新城疫强毒的攻击能１００％保护等特点,是一株良好的新城疫候选疫苗株