采用鸡胚尿囊腔接种培养鸡传染性喉气管炎弱毒疫苗的试验

采用鸡胚尿囊腔接种培养鸡传染性喉气管炎弱毒疫苗的试验王少英，赵丽明，陈任重，赵素霞（广东省生物药厂，广州．５１０６３０）鸡传染性喉气管炎病毒（ＬＴＶ）在鸡胚复制的位置主要在鸡胚绒毛尿囊膜（ＣＡＭ），传统的生产方法是采用在鸡胚做人工气室，通过人工气室接...     

鸡传染性喉气管炎活疫苗生产工艺的改进——鸡胚绒毛尿囊膜渗透法接种应用实验

采用鸡胚绒毛尿囊膜渗透法给鸡胚接种病毒,收获感染的鸡胚绒毛尿囊膜制备鸡传染性喉气管炎活疫苗,相比于传统的人工气室绒毛尿囊膜接种法和尿囊腔接种法可降低鸡胚的早死亡率,提高鸡胚利用率,降低生产成本,提高单产量。     

鸡传染性喉气管炎弱毒活疫苗生产工艺的优化及改进试验

《中华人民共和国兽用生物制品规程》规定,在生产鸡传染性喉气管炎弱毒活疫苗时,采用鸡胚尿囊腔接种和人工气室绒毛尿囊膜接种工艺生产成品。由于人工气室绒毛尿囊膜接种早死胚多,而且工艺比较繁琐,所以我国大多数生产企业在生产中多采用尿囊腔接种方法,但尿囊腔接种后病毒繁殖时间要比人工气室绒毛尿囊膜接种后延长1~2 d,增加了孵化成本,而且绒毛尿囊膜病变(增厚、水肿等)也不如人工气室绒毛尿囊膜接种后产生的病变严重(膜重)。为此,借鉴了鸡痘鹌鹑化弱毒活疫苗的新生产工艺———鸡胚绒毛尿囊膜渗透法[1],并将此方法用于鸡传染性喉气管炎…     

鸽痘病毒在鸡胚上的增殖和纯化研究

通过实验建立了鸽痘病毒的鸡胚绒毛尿囊膜和鸡胚尿囊腔培养方法，并对这两种方法的病毒收获量进行了比较研究。结果表明：这两种培养方法实用易行，实验所收集的病毒可以满足电子显微镜观察、核酸分析等科研及疫苗生产的需要。为鸽痘病毒的深入研究提供有利的试材。     

鸽痘病毒在鸡胚上的增殖和纯化研究

通过实验建立了鸽痘病毒的鸡胚绒毛尿囊膜和鸡胚尿囊腔培养方法,并对这两种方法的病毒收获量进行了比较研究.结果表明:这两种培养方法实用易行,实验所收集的病毒可以满足电子显微镜观察、核酸分析等科研及疫苗生产的需要.为鸽痘病毒的深入研究提供有利的试材.     

鸡传染性喉气管炎病毒培养特性的研究

本试验从发病鸡群分离一株鸡传染性喉气管炎病毒(命名为NM98a)，并用分离株和ILT王岗株对鸡胚绒毛尿囊膜、鸡胚尿囊腔及鸡胚肾细胞上培养进行比较研究。结果表明：ILTV经不同途径接种鸡胚和鸡胚肾细胞上培养，在鸡胚绒毛尿囊膜和鸡胚肾细胞上引起的病变不同，但最终增殖达到的感染性病毒量没有明显区别。鸡胚尿囊腔接种是ILTV增殖最佳途径。     

鸡传染性喉气管炎弱毒疫苗免疫异常的分析

鸡传染性喉气管炎(ILT)弱毒疫苗系用鸡传染性喉气管炎病毒弱毒株接种SPF鸡胚，收获感染鸡胚的绒毛尿囊膜，混合研磨，加适宜稳定剂，经冻干制成。该苗用于预防鸡传染性喉气管炎。     

鸡传染性喉气管炎弱毒疫苗生产工艺改进试验

鸡传染性喉气管炎（ILT）是由疱疹病毒科喉气管炎病毒引起的鸡的一种急性呼吸道传染病，我国已有许多地区爆发流行，给养鸡业带来了严重危害。通过疫苗接种可以有效防止本病的爆发和流行。我国使用的国产鸡传染性喉气管炎弱毒疫苗基本上都是采用鸡胚绒毛尿囊膜接种鸡胚，收获感染的鸡胚绒毛尿囊膜，混合研磨，加适当的稳定剂，经冷冻真空干燥制成。但是这种方法要制造人工气室，操作难度大，并且接毒后死胚率较高。为了简化生产工艺、降低生产成本，在不影响产品质量的前提下，我们采用直接接种尿囊腔培养病毒的方法，生产出几批传染性喉气管炎弱毒疫苗，质量检测达到《兽用生物制品质量标准汇编》（以下简称标准》）要求，并且使用效果较好，现将试验过程报告如下。     

鸡传染性法氏囊病毒增殖和纯化方法的研究

本试验通过IBDV标准毒株(IBDVBC6/85F4)和IBDV河北分离株分别接种于SPF鸡胚以及鸡胚成纤维细胞(CEF)。吸取接毒后2～5天内死亡鸡胚的尿囊液和绒毛尿囊膜,及细胞病变达到80%时的细胞培养液。经反复冻融,利用氯仿沉淀病毒,聚乙二醇6000浓缩病毒,蔗糖梯密度低温超速离心和葡聚糖G-200凝胶柱过滤等方法纯化病毒。经分光光度计检测,病毒的蛋白含量为1.377毫克/毫升,琼脂扩散试验证实被检抗原为IBDV。     

鸡传染性喉气管炎病毒和肾型支气管炎病毒同胚增殖的研究

将鸡传染性喉气管炎(ILT)病毒悬液和鸡传染性支气管炎(IB)病毒悬液，采取不同浓度，不同时间在同一个鸡胚上进行接种，可使两种病毒之间互不干扰，并能在同一鸡胚上共同增殖。取鸡胚绒毛尿囊膜检测传染性喉气管炎病毒(ILTV)，其EID50为10^55/0.2ml，取尿囊液检测传染性支气管炎病毒(IBV)，其EID50为10^63/0．2ml。与单项接种对照组相同；用同胚增殖的绒毛尿囊膜和尿囊液按一定比例混合，制造二联油乳剂灭活苗，免疫后30d和6个月后分别用ILT、IB强毒进行攻毒试验，结果免疫后30d，ILT和IB的保护率均为100％，免疫后6个月，ILT和IB的保护率分别为95.3％、86．7％。本试验结果表明，用此方法增殖病毒，既降低了成本，又保证了免疫效果，为今后制造二联苗提供了科学的依据。     

The Application and Research Progress of Chicken Embryo and Avian Cell Lines in Biomedicine

Chicken embryo has long been an important experimental model in basic and applied science because of its clear development process,especially in the early development of chicken embryo chorioallantoic membrane stage,due to its abundant blood vessels,it is a natural immunodeficiency host and can be used as an ideal experimental model for pathology,pharmacology and oncology research.The authors briefly described the tissue structure of early stage of chicken embryo,the application of chick embryo chorioallantoic membrane model in tumor,angiogenesis,organ transplantation,burn and other diseases,and the application of anti-cancer drug screening based on the pathological model of chicken embryo.The advances in the application of chicken embryo and avian cell lines in virus reproduction,vaccine production,therapeutic protein production and monoclonal antibody production were reviewed.A variety of human viruses,avian viruses and mycoplasma can proliferate in chicken embryos and avian cell lines and be used in vaccine production.In this paper,the development and characteristics of commonly used avian fibroblasts and pluripotent stem cells were described,and the source of commercial avian cell lines and some susceptible viruses were summarized.Chicken embryo expression system can produce human glycosylates at specific sites of target proteins,reduce the allergic reaction of the target protein to human,and poultry eggs are cheap and easily available,so it can be used as a suitable donor for the production of human monoclonal antibodies and therapeutic proteins.In this paper,the recent progress in the application of chicken embryos and avian cell lines in the field of biomedicine was introduced,and the future application of chicken embryos as animal models was forecasted.     

鸡全胚成纤维细胞在鸡痘细胞活疫苗生产中的应用

采用鸡胚全部组织(全胚法)制备鸡胚成纤维单层细胞和采用鸡胚部分组织(常规法)制备鸡胚成纤维单层细胞生产了鸡痘细胞活疫苗,接毒后均产生大量的感染多核细胞和典型细胞融合性病变(胡椒粒样).鸡胚效价检测表明,鸡痘细胞苗和鸡痘组织苗均可引起鸡胚绒毛尿囊膜水肿、增厚或痘斑,但鸡痘细胞苗产生痘斑数量明显高于鸡痘组织苗.鸡体刺种结果表明,鸡痘细胞苗诱发的免疫反应(发痘)好于鸡痘组织苗;与常规法相比,全胚成纤维单层细胞制备方法可提高鸡胚利用率2倍以上.全胚法也可应用于制备其他鸡成纤维细胞疫苗.     

THE ISOLATION OF LENTOGENIC STRAINS OF NEWCASTLE DISEASE VIRUS IN AUSTRALIA

SUMMARY Twelve isolations of Newcastle disease virus were made from 77 clinical samples from chickens with conjunctivitis, respiratory disease, proventriculitis and bursal atrophy. Nine of the Isolations were made from chickens with conjunctivitis. The viruses were identified as Newcastle disease virus by inhibition of their haemagglutinins with specific antiserum to Newcastle disease virus. The viruses failed to kill chicken embryos after inoculation into the allantoic cavity and they were judged to be lentogenic strains. There was no evidence that the Newcastle disease viruses were responsible for any of the clinical conditions from which they were isolated. The presence of other agents in 10 of the samples was indicated by reduced production of haemagglutinin in allantoic fluids of infected embryos, by deaths of infected embryos, by the production of cytopathic changes in avian cell cultures and by electron microscopy. Three isolations of infectious bronchitis virus, 2 of avian adenovirus and one of avian reovirus were made. Other samples were suspected of containing infectious bronchitis virus and mycoplasmas, but these were not isolated. The Newcastle disease viruses failed to produce plaques in chicken embryo fibroblast cell cultures and they were separated from the contaminating agents by haemagglutination and elution followed by passage at terminal dilution in chick embryos. No Newcastle disease virus was isolated from 60 caecal tonsils and 60 lung samples from 9-week-old broiler chickens. Eight lung samples yielded mycoplasmas that caused haemadsorption in chicken cell cultures. The mycoplasmas were probably Mycoplasma gallisepticum.     

Infectious tenosynovitis in broilers and broiler breeders in Egypt

Two infectious tenosynovitis-producing viruses were isolated from tendon sheaths and synovial fluids of 59 broilers and 15 broiler breeders obtained from different flocks in Egypt during June to October 1983. The viruses grew well on the chorioallantoic membrane of developing chicken embryos, produced small localized white pock lesions with oedematous swellings at the inoculation sites and death of most of the embryos 72 to 96 hours post-inoculation. They also induced cytopathic effect in chicken embryo rough, Vero and MS cell lines. The viruses were neutralized by reovirus S1133 antiserum, both in tissue culture and. on the chorioallantoic membrane. Inoculation of the viruses into 2-day-old broiler chicks via the foot pad, intramuscular and oral routes reproduced the disease with the development of characteristic clinical, pathological and serological responses. The infection was transmitted to in-contact control chicks. This is the first report of the disease and of the isolation and identification of the causative virus in Egypt.     

Infectious tenosynovitis in broilers and broiler breeders in Egypt

Two infectious tenosynovitis-producing viruses were isolated from tendon sheaths and synovial fluids of 59 broilers and 15 broiler breeders obtained from different flocks in Egypt during June to October 1983. The viruses grew well on the chorioallantoic membrane of developing chicken embryos, produced small localized white pock lesions with oedematous swellings at the inoculation sites and death of most of the embryos 72 to 96 hours post-inoculation. They also induced cytopathic effect in chicken embryo rough, Vero and MS cell lines. The viruses were neutralized by reovirus S1133 antiserum, both in tissue culture and on the chorioallantoic membrane. Inoculation of the viruses into 2-day-old broiler chicks via the foot pad, intramuscular and oral routes reproduced the disease with the development of characteristic clinical, pathological and serological responses. The infection was transmitted to in-contact control chicks. This is the first report of the disease and of the isolation and identification of the causative virus in Eqypt.     

The method of chicken whole embryo culture using the eggshell windowing,surrogate eggshell and ex ovo culture system

1. The unique accessibility of the avian embryo have made them an ideal model for the study of development and genome editing. Chicken whole embryo culture has provided important insights into toxicity tests, gene manipulation, clarifying gene functions, cell transplantation and cell tracking.

2. A simple technique for chicken manipulation is eggshell windowing, without or with seal, the latter having demonstrated some improvement in hatching rates.

3. Likewise, a surrogate eggshell system provides an accessible model for manipulation during chicken and quail development, with a higher hatchability compared to the simple windowing method.

4. The development of the chicken ex ovo culture systems in a synthetic environment as an efficient technique for imaging and microsurgery applications has enabled the study of important events of live chicken embryos at a specific time point.

5. This short review illustrates recent applications of well-designed whole embryo culture systems as a robust model for research into numerous biological mechanism, drug discovery, gene manipulating and production of functional proteins.     

新城疫、禽流感病毒同胚接种的研究

根据新城疫病毒、禽流感病毒两种病毒均能在鸡胚中繁殖的特点,将两种病毒以最适稀释比例稀释后,分别接种于9—11日龄的鸡胚尿囊腔,同时进行两种病毒同胚培养,并利用血凝试验进行病毒效价的测定。结果表明,在同一鸡胚中两种病毒均能较好地增殖,病毒间干扰现象不明显。本试验在增强疫苗的免疫原性和进一步阐明病毒的增殖机理方面具有重要意义。     

鸡痘病毒诱发细胞融合现象的观察

鸡痘病毒在鸡胚绒毛作囊膜及鸡胚成纤维细胞上交替传代后，用本试验提供的条件，可以明显诱发鸡胚成纤维细胞的融合，形成多量多核细胞。经鸡胚及实验鸡回归试验证实，这种细胞融合现象确系鸡痘病毒所为。     

Development of avian embryo manipulation techniques and their application to germ cell manipulation

The development of chicken embryo culture techniques, from single‐cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro. Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single‐cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non‐viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary.     

Inhibition of in vitro lymphocyte blastogenesis by chicken alpha-fetoprotein

Chicken alpha-fetoprotein (ch-AFP), purified from fetal chicken serum and embryo extracts, respectively, was examined for its immunomodulatory effect in vitro. Significant (P less than 0.05) suppression of the allogeneic mixed lymphocyte reaction (MLR) was observed, when these preparations were added to one-way mixed lymphocyte cultures (MLC) in quantities of 62.5-1000 micrograms/ml. Suppression of the MLR was depending on the presence of ch-AFP for at least 16 h after initiation of the MLC, suggesting that this fetal protein was acting mainly in the early phase of lymphoblastogenesis. Serum of chicken embryos (12th and 15th day of incubation), day-old chickens, and of 10-week-old chickens of four different inbred lines were also found to exert suppression of the MLR. From these data, it is hypothesized that ch-AFP plays an immunoregulatory role by maintaining a certain stage of self tolerance during differentiation of the avian immune system.