鸡痘病毒活疫苗污染禽网状内皮组织 增生症病毒的间接免疫荧光法检测研究

通过向无外源病毒污染的鸡痘病毒活疫苗中添加不同剂量的禽网状内皮组织增生症病毒(REV),然后用间接免疫荧光法(IFA)进行检测,确定了该IFA方法的最低检出量为每500羽份疫苗中污染20 TCID_(50)的REV。使用该方法对国内16家企业生产的60批鸡痘病毒活疫苗进行了检验,结果显示2个企业生产的4批疫苗REV检测为阳性。随机选取5批IFA检测阴性样品和4批IFA检测阳性样品,按鸡检查法进行外源病毒检验,结果两种方法对REV污染的检测结果的符合率为100%。     

鸡马立克病活疫苗中污染禽网状内皮组织增生症病毒检测方法的建立

为建立鸡马立克病(MD)活疫苗中污染禽网状内皮组织增生症病毒(REV)的间接免疫荧光检测方法,本研究通过向MD活疫苗中添加不同剂量的REV,用拟定的方法对样品检测REV。结果表明,Ⅰ型MD活疫苗、火鸡疱疹病毒活疫苗(Fc-126株)和MD二价活疫苗(HVT+CVI988株)中污染REV的最低检出量分别为每500羽份疫苗中污染5TCID50、10TCID50和15TCID50的REV。应用建立的方法对国内10家企业生产的43批MD活疫苗进行了检验,结果显示,2个企业生产的4批疫苗REV检测阳性。随机选取了5批IFA检测阴性样品和4批IFA检测阳性样品,按鸡检查法进行外源病毒检验,结果两种方法对REV污染检验结果的符合率为100%。因此,本研究建立的IFA法可替代鸡检查法,用于疫苗中REV污染的检验。     

部分禽源生物制品外源病毒的检测

进行了部分禽用生物制品外源病毒检测的鸡胚检查法、细胞检查法和鸡检查法。鸡胚检查法应用SPF鸡胚检验;细胞检查法主要通过鸡红细胞吸附试验、禽白血病病毒ELISA和禽网状内皮组织增生症病毒IFA检验疫苗是否含有外源鸡红细胞吸附因子、禽白血病病毒和禽网状内皮组织增生症病毒,并提出疫苗检验的判定标准,为各兽用生物制品生产企业新城疫疫苗的外源病毒检验提供参考。     

浅析禽用活毒疫苗污染外源病毒问题

疫苗的预防接种是目前有效控制家禽疫病的重要手段之一。然而被污染的疫苗不仅不能控制疾病,反而会加速疾病的传播。在许多地方,现在仍然有一些疾病通过污染的疫苗进行传播。由于疫苗生产原材料如组织、细胞基质、血清和胰蛋白酶等来源于动物,因此疫苗产品存在外源病毒污染的风险。通过分析过去禽用活毒疫苗污染外源病毒的事件,发现禽用活毒疫苗中污染禽白血病病毒、网状内皮组织增生症病毒和鸡传染性贫血病毒较为多见。本文就禽用活毒疫苗污染外源病毒产生的危害、历史与现状及疫苗外源病毒检测技术的优点和局限性等进行了综述,并提出了减少禽用活毒苗外源病毒污染的建议措施。     

不同消毒方式对禽用胚苗、种胚孵化率及疫苗质量的影响

禽用胚苗,系采用鸡胚、鸭胚等作为主要原材料生产的禽用疫苗.由于鸡胚产量大,供应足,价格低,取材广泛,生产周期短等优点,大多数禽用胚苗采用鸡胚作为主要原料.禽用病毒疫苗的生产,一般是以无特定免疫原性的鸡胚作为病毒繁殖的寄主,经孵化病毒扩增后,收获胚内病毒含量较高的部分用来制造疫苗.可见,在疫苗生产过程中需要用到大量的鸡胚.鸡胚成活率、外源物质对终产品的影响,是决定疫苗生产成本及品质的两个关键因素.其中,影响鸡胚成活率的一个重要原因是鸡胚外表面的细菌污染.为了寻找更适合实际生产的鸡胚外表面灭菌方法,提高鸡胚孵化成功率,降低生产成本,我们设计了如下试验:采用连续80余批次种胚,采用不同的消毒方式对鸡胚外表面进行处理,取得比较明显的效果.现报告如下:     

部分种鸡场常用活疫苗中ALV的PCR检测

禽白血病病毒(Avian leukosis virus,ALV)既能通过种蛋垂直传播给后代,也能通过鸡之间直接接触发生水平传播,而使用污染了ALV的活疫苗也是一种常见的传播途径。本课题组应用PCR方法,对3个种鸡场2年来使用的包括由10家疫苗公司生产的11种类型共计18份活疫苗样品进行了ALV的检测。结果有两份样品的检测呈阳性(占11.12%),它们分别是鸡传染性支气管炎疫苗和鸡痘疫苗,病毒亚型的鉴定结果显示,它们分别属于ALV的J和A亚型,而B、C和D亚型均未从样品中检出。本试验的结果表明,目前部分种鸡场使用的活疫苗中存在J和A亚型ALV的污染。这提示我们,疫苗污染可能是目前ALV在鸡群中传播的重要途径之一,应引起种鸡场的重视。     

Ⅰ群禽腺病毒疫苗研究进展

近年来,Ⅰ群禽腺病毒在多国引起家禽暴发疫情,主要表现为血清4和8型引起的心包积水-肝炎综合征和鸡包涵体肝炎,3~8周龄肉仔鸡高死亡率给养殖业造成了严重的经济损失。目前,我国国内尚无商品化Ⅰ群禽腺病毒疫苗,国内外有相关疫苗研制的很多报道,如灭活疫苗、减毒活疫苗及新兴起的基因工程疫苗,均能提供不同程度的免疫保护。文章综述了Ⅰ群禽腺病毒疫苗的国内外研究现状,以期为相关疫病的预防提供参考。     

关注我国SPF鸡胚生产——SPF鸡的概念

今年1月1日起,农业部对GMP疫苗生产企业疫苗菌（毒）种制备与鉴定、活疫苗生产以及疫苗检验使用无特定病原体（SPF级）鸡、鸡胚情况进行全面监督检查。对达不到标准要求的,将根据《兽药管理条例》规定进行处理。这就意味着我国对SPF种蛋生产企业、禽用活疫苗生产企业及其使用单位强制实施SPF化管理,兽药行业监管部门将加强禽用活疫苗生产SPF化的监督工作,全力推进禽用活疫苗生产的SPF化进程。     

禽源生物制品生产中蛋传性病原的控制方法

1 禽用活疫苗外源病毒检测 《中华人民共和国兽药典》(2010年版,三部)对活疫苗外源病毒检测的要求: 1.1 样品处理 取样品2～3瓶,对毒种或活疫苗进行检验时,将样品混合,用相应有特异性抗血清中和后作为检品(另有规定除外);对细胞进行检验时,经3次冻融后混合作为检品;用鸡检查法检验时,样品不处理.     

家禽活疫苗中禽白血病病毒污染检测方法比较

本研究对家禽活疫苗中禽白血病病毒的不同检测方法进行了对比,通过直接ELISA检测法、RT-PCR法、病毒分离法和SPF鸡抗体检测法,对不同来源的家禽活疫苗进行了禽白血病病毒污染的检测。通过进一步测序验证发现,直接ELISA检测法和RT-PCR检测法都可出现"假阳性"。试验结果表明,病毒分离法虽然结果准确,但是操作难度大、技术要求高,而SPF鸡抗体检测法既简单、准确,又能确切反映疫苗的纯净性,利于推广应用。     

Detection of avian adenovirus by polymerase chain reaction

An avian adenovirus-specific polymerase chain reaction was developed. The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome. An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III. The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria. As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis.     

禽白血病-肉瘤病毒的RT-PCR检测

应用RT-PCR对2株禽白血病病毒（ALV）和1株劳斯肉瘤病毒（RSV）进行了检测试验.试验提取了病毒RNA,并使用3对ALVgp85基因引物对其进行了反转录、扩增,从而建立了ALV-RSV RT-PCR.使用无相关性的禽RNA和DNA病毒进行特异性试验和使用ALV进行的敏感性试验表明,该方法是一种快速、特异、敏感的体外试验,可用于禽源病毒种毒和疫苗中ALV和RSV的污染检测.     

2019年上海地区H9N2禽流感病毒分子特征及演化分析

为了解上海市活禽市场H9N2亚型禽流感病毒(AIV)分离株的遗传变异情况,本研究对2019年分离的4株H9N2AIV的8个基因节段进行PCR扩增、克隆和测序,并对获得的8个基因序列进行同源性以及基因进化分析,对与病毒适应性增加的关键氨基酸位点进行了分析,并和目前我国使用的H9N2流感疫苗毒株HA上的抗原位点进行了比较....     

Adenoviral pancreatitis in guinea fowl (Numida meleagris)

Necrotizing pancreatitis was observed in 2-week-old Guinea fowl submitted for necropsy and histopathology. Intranuclear inclusion bodies seen histologically in acinar epithelium were examined by electron microscopy and found to contain viruses resembling adenovirus. Adenovirus was isolated in embryonated eggs from the pancreata of affected birds. The adenovirus isolated was not neutralized by chicken antisera developed against 10 known serotypes of group 1 avian adenoviruses.     

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.     

鸡胚及禽类细胞系在生物医药领域的应用及研究进展

鸡胚因发育过程清楚，长久以来作为基础和应用科学研究领域重要的实验模型，尤其在鸡胚发育早期绒毛尿囊膜阶段，因其血管丰富，是天然的免疫缺陷宿主，是病理学、药理学和肿瘤学等研究领域的理想实验模型。作者简述了发育早期的鸡胚组织结构，并介绍了鸡胚绒毛尿囊膜在肿瘤研究、血管生成、器官移植、烧伤等疾病机理研究中的应用，以及在鸡胚病理模型基础上进行的抗肿瘤药物筛选的应用。重点介绍了鸡胚及禽类细胞系在病毒繁殖和疫苗生产、治疗性蛋白和单抗生产方面的应用研究进展。多种人源病毒、禽源病毒、支原体等可在鸡胚及禽类细胞上增殖，并用于疫苗生产。作者对常用的禽类纤维原细胞和多能干细胞的发展和特点进行了阐述，并总结了商业化的禽类细胞系来源以及部分易感病毒。鸡胚表达系统能够在目的蛋白特定位点产生人源化糖基，减少目的蛋白对人的过敏反应，且禽蛋廉价易得，可作为生产人用单克隆抗体和治疗性蛋白的合适供体。作者介绍了鸡胚及禽类细胞系在生物医药领域应用的最新进展，并对鸡胚作为动物模型在未来的应用进行了展望。     

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.     

Genomic identification and characterization of avian adenoviruses associated with inclusion body hepatitis.

Four pathogenic avian adenovirus isolates associated with inclusion body hepatitis and mortality in commercial broiler chicks and chickens were characterized and identified. These group I avian adenovirus isolates were classified as group E (serotypes 6, 7, 8, and 9) avian adenoviruses on the basis of the restriction enzyme patterns of their viral DNA. Isolate 3718 was neutralized by a serotype 6 reference avian adenovirus antiserum and isolates 8193, 8380, and 8565 were all neutralized by a serotype 8 reference avian adenovirus antiserum by virus neutralization assays. Infectivity and virulence such as mortality, hemorrhages, enlarged green livers with intranuclear inclusion bodies, stunting, intestinal sloughing, and poor feathering were observed in specific-pathogen-free chicken embryos and were identical for all four isolates when embryos were inoculated via the yolk sac and/or chorioallantoic membrane. Complete mortality was observed within 72 hr postinoculation in specific-pathogen-free (SPF) chickens inoculated intramuscularly for all four avian adenovirus isolates.     

Adenovirus pathogenicity in immature ostriches.

Two group I avian adenoviruses implicated as the possible cause of "fading chick syndrome" in ostriches less than 8 wk of age were isolated in primary chicken embryo liver cells. These viruses were identified by virus neutralization and further characterized by a pathogenicity trial in immature ostriches. The results showed that these isolates were noninfectious in ostrich chicks.     

4型禽腺病毒HLJ1701株灭活疫苗的研制

利用4型禽腺病毒HLJ1701株进行灭活疫苗的研制,并对疫苗的免疫效果进行评价,为家禽4型禽腺病毒的防控提供数据及参考。将HLJ1701株用灭菌生理盐水作10~4倍稀释后,接种9日龄SPF鸡胚,37℃孵育72 h后收获感染鸡胚尿囊液,经甲醛灭活后,加白油佐剂乳化制成油乳剂灭活疫苗,对制备疫苗的性状、安全性、免疫效力等进行检验。结果显示,制备的3批4型禽腺病毒灭活疫苗(HLJ1701株)均为油包水型,黏度均在50 cP以内,对3批疫苗取样,样品经3000 r/min离心15 min,管底无水相析出。安全性试验结果显示,将疫苗按1 mL/只超剂量接种3周龄SPF鸡,试验鸡在观察期内全部健活,未出现局部或全身不良反应,表明疫苗对SPF鸡具有良好的安全性;免疫效力及攻毒保护试验结果显示,用疫苗按0.2 mL/只的剂量免疫接种3周龄SPF鸡1次,免疫接种后21d试验鸡血清中HLJ1701株的抗体平均效价可达2~8以上,使用4型禽腺病毒(HLJ1701株)接种0.2 mL/只(100 LD_(50))对免疫鸡进行攻毒,疫苗对免疫鸡的保护率均为100%。研究表明,实验室条件下研制的4型禽腺病毒(HLJ1701株)灭活疫苗的各项指标均符合标准。