禽核酸疫苗的研究进展

本文对禽用（鸡、鸭、火鸡）核酸疫苗的研究进行综述。首先描述禽用核酸疫苗的进展：病原,质粒以及免疫途径。其次,描述提高核酸疫苗免疫效果的方式：接种途径,疫苗剂量以及首免时间,增加宿主细胞对质粒的摄入,添加免疫增强分子,优化质粒骨架和密码子,疫苗抗原的选择,异源性的首免-加强免疫策略。最后,描述禽用核酸疫苗的其他特点：接种后质粒的去向,免疫反应的特点以及核酸疫苗的其他用途。     

奶牛免疫布鲁氏菌A19疫苗后的抗体变化和排菌情况监测

为探究奶牛免疫布鲁氏菌A19疫苗（以下简称A19疫苗）后的抗体变化规律及向外排菌的风险,使用A19疫苗免疫30头3月龄奶牛,在首免和加强免疫后3个月内,通过虎红平板凝集试验（RBT）和试管凝集试验（SAT）进行抗体检测,采用荧光定量PCR方法,进行血液以及口鼻、阴道拭子的布鲁氏菌核酸检测。结果显示：首免后6 d,奶牛抗体开始出现阳转,60 d后绝大部分奶牛抗体转阴,而加强免疫后3 d,又出现抗体阳转;仅在首免后2 d内的阴道拭子和首免后6 d内的血液中检测到布鲁氏菌核酸,而在口鼻拭子中以及其他时间点的血液和阴道拭子中均未检测到布鲁氏菌核酸。结果表明,A19疫苗免疫奶牛抗体阳转快,排菌期短,安全性高。     

肠炎沙门菌疫苗株Sm24/Rif12/Ssq在蛋鸡体内的定殖规律

为明确肠炎沙门菌疫苗株Sm24/Rif12/Ssq在蛋鸡体内的定殖规律,选取50只海兰褐蛋鸡,于0日龄(出雏当日)滴口免疫1羽份(1×10⁸ CFU/只)Sm24/Rif12/Ssq疫苗(首免),分别于免疫后1、3、5、7d采集咽拭子、泄殖腔拭子以及盲肠和肝脏样品测定疫苗株定殖水平,并于免疫后30 d采集血液测定抗体水平;随后选取30只经过首免的6周龄蛋鸡和30只经过二免的16周龄蛋鸡,分别滴口免疫1羽份(1×10⁸ CFU/只)Sm24/Rif12/Ssq疫苗,即为二免和三免,于免疫后4 d采集泄殖腔拭子,测定疫苗株定殖水平,并于免疫后30、60、100 d采集血液测定抗体水平。结果显示,首免后咽拭子、泄殖腔拭子和盲肠样品中疫苗株载量呈先上升后下降趋势,疫苗株核酸检出率60%～100%,肝脏样品中疫苗株载量呈逐步下降趋势,疫苗株核酸检出率50%-90%,但未从血液样品中检出抗体;二免和三免后泄殖腔拭子中疫苗株核酸检出率为17%～20%,血清抗体阳性率呈逐步上升趋势。结果表明,Sm24/Rif12/Ssq疫苗株易定殖于0日龄蛋鸡肠道,而较难定...     

猪传染性胃肠炎核酸疫苗免疫小鼠后的血清抗体IgG检测

本试验旨在对猪传染性胃肠炎核酸疫苗(含有TGEV-S基因质粒)开展免疫原性研究,为科学评价该TGE核酸疫苗的免疫效果提供依据。通过灌胃口服免疫TGE核酸疫苗诱导小鼠机体产生特异性免疫,定期采样监测其血清抗体产生规律,采用间接ELISA方法测定免疫小鼠血清抗体IgG的抗体效价。结果发现:TGE核酸疫苗免疫小鼠后有效地激发了TGEV特异性抗体,首免后第二周开始就能检测到血清抗体应答,且该组抗体水平一直都与空载体组和PBS空白对照组之间呈极显著差异(P0.01),表现出良好的免疫原性。     

鸡新城疫Ⅰ系疫苗对雏鸡初次免疫试验

长期以来,肉鸡新城疫的免疫程序一直是沿用先进行Ⅱ系疫苗首免,再用Ⅱ系或Ⅰ系疫苗二免的方法。近年来,我们在鸡新城疫免疫程序的研究和实践中感到,鸡新城疫Ⅱ系疫苗首免和Ⅰ系疫苗再免之间免疫空档期长,而且Ⅱ系疫苗对雏鸡初次免疫效果不够确实,产生免疫应答时间长,免疫期短,因而雏鸡应用现行免疫程序免疫,仍屡有新城疫发生,使肉鸡生产造成很大损失。为解决这一生产中的难     

猪伪狂犬首免日龄的确定

<正>自2011年猪伪狂犬病在国内猪场大面积流行以来,伪狂犬的控制和净化成为大多数公司和养殖户关心的问题。众所周知,疫苗免疫是预防伪狂犬疾病最为有效的方式,而制定出合理有效的免疫方案尤其是首免日龄的确定对于伪狂犬的防控至关重要。目前,国内外有多种可供选择的疫苗生产厂家,由于各厂家疫苗抗原含量不同,母源抗体持续时间及猪群健康状况不同,首免日龄亦不同。场区应根据本场的具体情况确定首免日龄。     

高致病性猪蓝耳病弱毒疫苗与灭活苗免疫效果评估

相同饲养条件下的35日龄仔猪分别使用高致病性猪蓝耳病弱毒疫苗与灭活苗进行首免,并分别在首免当日、首免后28 d、首免后58 d检测高致病性猪蓝耳病免疫抗体水平。结果发现,使用弱毒疫苗免疫的猪高致病性猪蓝耳病抗体上升速度和抗体水平均优于使用灭活苗免疫猪。试验结果表明,高致病性猪蓝耳病弱毒疫苗可在接种后21-28 d产生免疫力,免疫期为4个月,猪繁殖与呼吸综合征流行的地区应首先考虑使用弱毒疫苗进行免疫。     

鸡新城疫免疫程序的研究

通过对75只雏鸡的母源抗体及免疫效果检测表明,首免成功后20天,各组雏鸡 HI 抗体均由 log_25以上下降到 log_24以下。此时再用Ⅱ系疫苗作2、3次重复免疫,没有效果,用Ⅰ系疫苗肌注2免,效果确实,无副作用。因此,在不具备免疫检测条件的情况下,建议在种鸡免疫效果确实的养鸡场、专业户采用15～20日龄Ⅱ系疫苗滴鼻首免,40～59日龄Ⅰ系疫苗肌注2免;对于种鸡未免疫或免疫时间过长的个体户、专业户,可采用1～4日龄Ⅱ系疫苗首免,15～20日龄Ⅱ系疫苗2免,40～50日龄用Ⅰ系疫苗肌注免疫。     

不同免疫程序对仔猪接种猪传染性胸膜肺炎疫苗免疫效果的影响

为规模猪场制定科学合理的猪传染性胸膜肺炎免疫程序提供依据,试验研究了不同免疫程序对仔猪接种猪传染性胸膜肺炎疫苗免疫效果的影响。结果显示,仔猪经不同免疫程序接种猪传染性胸膜肺炎疫苗后,其免疫效果和增重存在差异。其中,30日龄首免、60日龄二免(G组)的猪传染性胸膜肺炎免疫程序的免疫效果最好,整个试验阶段未发现传染性胸膜肺炎感染猪,且该组猪的增重情况也较好,为112.15 kg;其次是35日龄首免、65日龄二免(A组),38日龄首免、68日龄二免(B组),35日龄首免、75日龄二免(D组),35日龄首免、70日龄二免(E组),35日龄首免、60日龄二免(F组);而42日龄首免、72日龄二免(C组)的免疫效果最差。     

河源某猪场两种猪瘟疫苗田间免疫效果对比试验

通过在河源某猪场,对不同厂家两种猪瘟疫苗单独使用及混合使用后的的免疫效果对比,得出以下结论：首免和二免均使用猪瘟传代细胞苗免疫的效果最好;首免使用猪瘟脾淋苗,二免使用猪瘟传代细胞苗免疫的效果次之;首免和二免均使用脾淋苗免疫的效果最差。     

减毒鼠伤寒沙门氏菌载体在兽医疫苗中的运用

重组疫苗的研究中,减毒鼠伤寒沙门氏菌可以作为真核表达载体。其原理是将免疫原基因片段连接到某种真核质粒中,然后将质粒导入减毒鼠伤寒沙门氏菌,构建针对该特异病原的重组活疫苗。本文介绍了减毒鼠伤寒沙门氏菌作为DNA载体,在动物细菌、病毒和支原体重组活疫苗中的运用。     

Immunomodular effect of fusion gene DNA vaccine of avian metapneumoviruses

The objective of this work was to develop and evaluate the immunomodular effect of a DNA vaccine based on the fusion (F) gene of avian metapneumoviruses (aMPV) and to study its protection against field virus challenge, as this will help to better control the disease in turkeys. In this study, the F protein of the Egyptian isolate (Giza-turkey rhinotracheitis-4) of the B-subtype of aMPV isolated in 2009 was expressed from a DNA plasmid in Vero cells. After 1 i.m. injection of turkey poults with this plasmid, the antibody response was detected by ELISA. The turkey poults inoculated with locally prepared DNA aMPV vaccine had highly significant phagocytic activity, as measured by phagocytic percent and index of macrophage activation, in comparison to those inoculated with inactivated and live attenuated vaccines and with the noninfected control group. Intratracheal challenge of turkey poults at 21 d postvaccination by a dose of 100 uL of field Egyptian Giza-turkey rhinotracheitis-4 virus of a titer 6 log₁₀ tissue culture infective dose 50 resulted in 100% protection in poults that received locally prepared DNA aMPV vaccine, whereas those that received commercial aMPV vaccines experienced 80 and 90% protection; typical clinical signs of aMPV infection were seen in control nonvaccinated poults. Therefore, a high success rate was noted when using F gene DNA plasmid vaccine by the induction of a potent immunomodular effect for both cell-mediated and humoral immune response. The use of the F gene DNA plasmid vaccine developed in this study provided 100% protection in vaccinated poults, which can help in controlling aMPV infections in turkeys.     

Potency of an experimental DNA vaccine against Aujeszky's disease in pigs

Intradermal vaccination with plasmid DNA encoding envelope glycoprotein C (gC) of pseudorabies virus (PrV) conferred protection of pigs against Aujeszky's disease when challenged with strain 75V19, but proved to be inadequate for protection against the highly virulent strain NIA-3. To improve the performance of the DNA vaccine, animals were vaccinated intradermally with a combination of plasmids expressing PrV glycoproteins gB, gC, gD, or gE under control of the major immediate-early promotor/enhancer of human cytomegalovirus. 12.5 microg per plasmid were used per immunization of 5-week old piglets which were injected three times at biweekly intervals. Five out of six animals survived a lethal challenge with strain NIA-3 without exhibiting central nervous signs, whereas all the control animals succumbed to the disease. This result shows the increased protection afforded by administration of the plasmid mixture over vaccination with a gC expressing plasmid alone. A comparative trial was performed using commercially available inactivated and modified-live vaccines and a mixture of plasmids expressing gB, gC, and gD. gE was omitted to conform with current eradication strategies based on gE-deleted vaccines. All six animals vaccinated with the live vaccine survived the lethal NIA-3 challenge without showing severe clinical signs. In contrast, five of six animals immunized with the inactivated vaccine died, as did two non-vaccinated controls. In this test, three of six animals vaccinated with the DNA vaccine survived without severe clinical signs, whereas three succumbed to the disease. Comparing weight reduction and virus excretion, the DNA vaccine also ranged between the inactivated and modified-live vaccines. Thus, administration of DNA constructs expressing different PrV glycoproteins was superior to an adjuvanted inactivated vaccine but less effective than an attenuated live vaccine in protection of pigs against PrV infection. Our data suggest a potential use of DNA vaccination in circumstances which do not allow administration of live attenuated vaccines.     

禽流感病毒H5HA、H7HA基因共表达核酸疫苗质粒的构建及其在体外的表达

为了获得既能对 H5亚型又能对 H7亚型 AIV攻击产生保护的核酸疫苗 ,设计构建了含有双顺反子的真核表达载体 ,用限制性内切酶从 PCIH7HA载体上切得 AIV H7HA基因的c DNA,此 c DNA含有 CMV启动子和 Poly(A)终止序列 ,使用 DNA 连接酶引入载体PCIH5HA中 ,最后获得双顺反子真核表达载体PCI-H5HA-H7HA,其中 H5HA和 H7HA基因都带有自己的 CMV启动子和 Poly(A)终止序列。选用 2 93 -T真核表达细胞系 ,用脂质体转染法导入细胞进行瞬时表达 ,转染后 48h用荧光标记的兔抗鸡 Ig G进行间接免疫荧光试验。结果表明双顺反子真核表达载体构建正确 ,且在真核表达细胞系中 H5HA和 H7HA基因都能获得表达 ,且 H5HA 基因的表达效果优于H7HA。这为进一步的动物免疫试验和疫苗效价检测试验奠定了基础     

DNA fingerprinting of plasmid-containing serotype A: 3,4 Pasteurella multocida isolated from cases of fowl cholera in chickens and turkeys

The live, attenuated vaccine strains of Pasteurella multocida have been hypothesized to be responsible for homologous serotype outbreaks of fowl cholera on farms that use the commercial vaccines. We have further hypothesized that the naturally occurring Clemson University (CU) vaccine strain may be transformed to virulence by the acquisition of plasmid DNA. To test this hypothesis, we obtained seven homologous serotype (A:3,4) P. multocida isolates, all plasmid bearing, that were cultured from fowl cholera cases in vaccinated flocks and compared the isolates with the CU reference vaccine by molecular methods. Restriction fragment length polymorphisms (RFLPs) were detected by DNA/DNA hybridization with labeled probes specific for the cya, aroA, and rrn genes of P. multocida. The RFLPs obtained from BglII-digested genomic DNA probed with cya demonstrated no differences among the isolates. Although three isolates probed with aroA showed a RFLP identical to the vaccine strain, five isolates were distinctly different. Isolates probed with rrn grouped into three different restriction patterns that were dissimilar from that of the vaccine strain. Therefore, we have shown that these fowl cholera isolates are different from the CU vaccine strain and that these outbreaks were not vaccine related.     

鸭瘟病毒转移质粒的构建

为了建立重组鸭瘟病毒技术,构建了鸭瘟病毒转移质粒。在对鸭瘟强毒和弱毒株TK基因进行测序分析后,将鸭瘟病毒TK-UL24DNA片段克隆于pUC18载体中,构建了质粒pTK;将PCR扩增的GFP真核表达盒插入pTK质粒的TK基因内部,获得转移载体质粒pTK-GFP。鸭瘟病毒TK-UL24测序分析表明鸭瘟强、弱毒株TK基因序列完全相同;转移载体携带Pcmv-GFP-SV40pA表达盒,测序验证其序列与源序列一致。pTK-GFP在脂质体介导下,转染鸭胚成纤维细胞和鸭肾细胞,在荧光显微镜下观察绿色荧光蛋白表达情况。质粒转染细胞后,绿色荧光蛋白得到了有效的表达,为进一步开展重组鸭瘟病毒的研究和构建具有遗传标记的鸭瘟疫苗奠定了基础。     

A recombinant DNA vaccine encoding C. andersoni oocyst wall protein induces immunity against experimental C. parvum infection

Cryptosporidium andersoni parasited in the abomasum has been demonstrated as a cause of reduction of milk production in dairy cow. In this study, a novel chimeric DNA vaccine pVAX1-AB was constructed and the efficacy against Cryptosporidium parvum was determined. BALB/c mice were divided into 3 groups and immunized with DNA vaccine expressing the oocyst wall protein, AB protein of C. andersoni, the recombinant plasmid containing the AB gene, respectively. After inoculation of 1 × 10(6) oocysts of C. parvum, the humoral and cellular immune responses were detected. Experimental results showed that the recombinant plasmid can induce corresponding specific antibody response, simultaneously influenced cellular immune responses, and provided greater protection rate (48.6%) than the other groups. These results indicated that chimeric DNA vaccine has a potential in Cryptosporidium vaccine development.     

A novel transcutaneous plasmid-dimethylsulfoxide delivery technique for avian nucleic acid immunization

In this report, we show that dimethylsulfoxide (DMSO) enhances liposome-mediated transfection of nucleic acid in chicken macrophage cells and that this could be exploited for the transcutaneous delivery of naked DNA through the intact skin of chickens. We found that DMSO enhanced transfection efficiencies of lipofectamine and polyethyleneimine in HD-11 chicken macrophage cells. Based on this principle, we showed that transcutaneous delivery of a DNA plasmid-dimethylsulfoxide mixture (1:1) to untreated skin of chickens results in a wide distribution of the plasmid in the body. Distribution studies were done using plasmids encoding enhanced green fluorescent protein (EGFP) reporter gene and a bivalent DNA vaccine coding for infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) immunogenic protein genes. This bivalent vaccine induced mucosal and systemic immune responses, as evidenced by IgA and IgM production in the tears and serum of vaccinated chickens. Mucosal immune responses in the tears after topical vaccination were significantly higher (P < 0.05) than after i.m. delivery of the same DNA vaccine and were characterized by the absence of an IgG response. The biodistribution of plasmid indicated that topical delivery with DMSO resulted in a wide distribution and persistence of the plasmid until 15 weeks post-primary vaccination. Both delivery methods resulted in insert-specific message being made in several body tissues, but after topical delivery the virus-specific mRNA could be detected in the bone marrow of one out of three chickens until 15 weeks post-primary vaccination. Furthermore, transcutaneous delivery of this DNA vaccine using DMSO conferred protection from challenge with virulent IBDV (86% survival) and NDV (86% survival). This novel transcutaneous method of delivery of a DNA vaccine shows promise as being an easy and effective way to deliver nucleic acids through intact skin for vaccination or therapeutic purposes.