外源病毒检验用抗鸭瘟病毒特异性阳性血清的制备与检定

为制备鸭瘟种毒和鸭瘟活疫苗检验用抗鸭瘟病毒特异性阳性血清,对6个月龄左右的山羊进行五次免疫。最后一次免疫后2周采血并分离血清,血清经混合、分装、冷冻真空干燥后,对其进行了无菌检验、外源病毒检验、剩余水分测定、中和效价测定、均匀性检验。结果表明,本研究制备的抗鸭瘟病毒阳性血清特异性良好,无菌检验、外源病毒检验和剩余水分测定均符合《中华人民共和国兽药典》(2010年版三部)规定;血清中和效价大于1∶100,均匀性良好,可满足兽药典标准中鸭瘟种毒和鸭瘟活疫苗的鉴别检验和外源病毒检验。     

鸭瘟活疫苗鸡胚效检与免疫攻毒保护的平行关系

鸭瘟(鸡胚化弱毒株)活疫苗采用鸡胚与易感鸭免疫攻毒的2种方法进行效力检验,通过试验证明当成品效价≥103.7ELD50/羽时,2种检验方法存在正相关。     

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%。     

禽用活疫苗外源病毒污染情况的调查研究

为对禽用活疫苗污染外源病毒的情况进行彻底摸底调查,抽取了目前使用量大、覆盖面广的禽用活疫苗重点品种共8种28批,涉及18家国内生产企业、7家国外企业,疫苗生产用SPF鸡胚来自国内8家企业。按照《中国兽药典》2010年版三部鸡检查法和《欧洲药典》对抽检的禽病活疫苗进行了外源病毒污染和禽腺病毒污染检验。结果显示所抽检的禽用活疫苗均无外源病毒污染和禽腺病毒污染,表明我国禽用活疫苗的质量控制和安全性良好。     

鸭瘟与鸭坦布苏病毒病活疫苗联合免疫效果研究

鸭瘟鸡胚化弱毒活疫苗与鸭坦布苏病毒病活疫苗通过肌肉注射途径,同时接种21日龄健康易感肉鸭,检测两种活疫苗联合免疫的安全性和免疫效力。将两种活疫苗同时免疫雏鸭,免疫后14d检测血清抗体值,并在14d时采用鸭瘟强毒和鸭坦布苏病毒强毒株同时攻毒,观察试验组与对照组的攻毒保护情况。结果表明:两种活疫苗联合免疫健康易感雏鸭后,血清抗体上升快,可同时抵御鸭瘟强毒与鸭坦布苏病毒强毒株的攻击,保护效果良好。结论:鸭瘟与鸭坦布苏病毒病活疫苗联合免疫,对雏鸭安全,免疫保护效果好,避免多次免疫对雏鸭带来的应激反应,提高养殖经济效益。     

肉食兽细小病毒阳性血清的制备与应用

为了研制用于检验肉食兽细小病毒相关制品的阳性血清，试验采用猫细小病毒(FPV)-A株对健康易感狐狸进行基础免疫，再用具有致病性的水貂肠炎病毒(MEV)-ZJ1株攻毒免疫的狐狸进行加强免疫，攻毒后第14天无菌采集血液，分离血清，并参照《中华人民共和国兽药典》中的方法对该血清进行细菌、支原体和外源病毒检验及中和抗体效价测定。通过观察细胞病变情况和进行免疫荧光检测测定中和指数，并用阳性血清对3个不同批次的水貂犬瘟热和细小病毒性肠炎二联活疫苗进行外源病毒检验和鉴别检验。结果表明：所制备的血清无细菌、支原体和外源病毒污染。对不同动物来源的肉食兽细小病毒均具有良好的中和作用，中和抗体效价为1∶2 330。对不同动物来源的肉食兽细小病毒的中和指数均大于1×10^4.7。3个批次的水貂犬瘟热和细小病毒性肠炎二联活疫苗顺利通过复合检验。说明试验制备的阳性血清能够用于肉食兽细小病毒相关生物制品的外源病毒检验和鉴别检验。     

一株鸭瘟病毒(GM株)的分离与鉴定

针对山东潍坊地区鸭瘟零星发病现象,从潍坊采集具有典型症状病料,通过鸭胚接种,分离出一株病毒。通过分子生物学以及阳性血清定性中和试验鉴定为鸭瘟病毒(DPV),命名为鸭瘟病毒GM株。结果显示,该本病毒可适应鸭胚和鸡胚成纤维细胞;鸭胚ELD50为10-5.5/0.2 m L;该病毒无血凝活性,不能凝集1%鸡红细胞;本动物回归,使樱桃谷鸭在5~6 d死亡,成功复制出鸭瘟病毒;鸭瘟病毒GM株与鸭瘟病毒鸡胚化弱毒株(CVCC AV1222)疫苗的免疫攻毒试验表明,传统鸭瘟疫苗对该分离株具有保护力。初步确定该病毒为传统鸭瘟野毒。     

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。     

外源病毒检验用抗鸡传染性法氏囊病病毒特异性血清的制备与检定

为制备外源病毒检验用抗鸡传染性法氏囊病病毒(Infectious Bursal Disease Virus,IBDV)的特异性血清,对300只3~4周龄的SPF鸡进行了基础免疫和加强免疫。最后一次免疫后21d采血并分离血清,血清经混合、分装、冷冻真空干燥后,对其进行了无菌检验、外源病毒检验、剩余水分测定、中和效价测定和特异性检验。结果表明,本研究制备的抗IBDV特异性血清无菌检验、外源病毒检验和剩余水分测定均符合《中华人民共和国兽药典》三部(二〇一〇年版)规定;血清中和效价为1:5747,与IBDV B87株毒种中和指数为104.3,与鸡新城疫病毒La Sota株、鸡传染性支气管炎病毒H120株、鸡传染性支气管炎病毒H52株、鸡传染性喉气管炎病毒、鸡痘病毒、禽呼肠孤病毒S1133株的中和指数均不大于10,通过AGP、ELISA、HI等试验确定血清中不含其他禽源外源病毒抗体。本研究为鸡传染性法氏囊病活疫苗或毒种的外源病毒检验提供了具有良好特异性和高效价的中和用血清。     

外源病毒检验用抗鸡传染性支气管炎病毒特异性血清的制备与检定

为制备外源病毒检验用抗鸡传染性支气管炎病毒(IBV)特异性血清,对300只3~4周龄的SPF鸡进行基础免疫和加强免疫;最后一次免疫21 d后,采血并分离血清;对血清经混合、分装、冷冻、真空干燥后,进行无菌检验、外源病毒检验、剩余水分测定、中和效价测定和特异性检验。结果表明:本研究制备的抗IBV特异性血清的无菌检验、外源病毒检验和剩余水分测定结果均符合现行《中华人民共和国兽药典》规定;血清中和效价为1:800,与IBV H120种毒株的中和指数为106.3,与鸡传染性法氏囊病毒B87株、鸡新城疫病毒La Sota株、鸡传染性喉气管炎病毒、鸡痘病毒、禽呼肠孤病毒S1133株的中和指数均不大于10;通过AGP、ELISA、HI等试验,确定该血清中不含其他禽源外源病毒抗体。本研究为鸡传染性支气管炎活疫苗或毒种的外源病毒检验提供了具有良好特异性和高效价的中和用血清。     

鸭瘟诊断方法研究进展

鸭瘟是由疱疹病毒引起的鸭、鹅的一种烈性传染病。目前已有多种诊断方法用于鸭瘟的诊断。本文就鸭瘟诊断方法的研究进展作一综述。     

A Plaque Assay for Duck Plague Virus

A plaque assay for duck plague virus was developed for a chicken embryo-adapted virus and a duck lethal virus and used to determine the identity of these viruses. Using the plaque inhibition neutralization test, duck plague virus was differentiated from Newcastle disease, fowl plague, and duck hepatitis viruses. The plaque morphology is described.     

Diagnosis of duck plague in waterfowl by polymerase chain reaction

A recently developed polymerase chain reaction (PCR) assay was used for diagnosis of duck plague in waterfowl tissues from past and current cases of waterfowl mortality and to identify duck plague virus in combined cloacal/oral-pharyngeal swab samples from healthy mallards (Anas platyrhynchos) after a disease outbreak. The PCR was able to detect viral DNA from all the individual or pooled tissues assayed from 10 waterfowl, including liver and spleen samples from three Muscovy ducks (Cairina moschata domesticus) that did not yield virus isolates. The strong staining intensity of the PCR products from the waterfowl tissues indicated that large amounts of virus were present, even when virus was not isolated. Duck plague DNA was also detected in a cloacal swab sample from a wood duck (Aix sponsa) carcass submitted for diagnosis. The PCR assay identified duck plague DNA in 13 swab samples that produced virus isolates from carrier mallards sampled in 1981 after a duck plague die-off. The duck plague PCR clearly demonstrated the ability to quickly diagnose duck plague in suspect mortality cases and to detect virus shed by carrier waterfowl.     

Identification of duck plague virus by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primers sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague.     

鸭圆环病毒巢式PCR检测方法的建立及应用

鸭圆环病毒病给养鸭业造成严重的经济损失,鉴于此,本研究根据GenBank登录的鸭圆环病毒(duck circovirus,DuCV)保守区基因组序列(登录号:NC_005053.1),设计合成内、外2对引物,通过PCR反应条件的优化,建立了检测DuCV的巢式PCR检测方法。该方法对鸭病毒性肝炎病毒、鸭瘟病毒、鸭细小病毒、新城疫病毒的扩增结果均为阴性;第1轮扩增的灵敏度为10 pg,第2轮扩增的灵敏度为0.1 pg,通过巢式PCR,敏感性提高了100倍。本研究建立的巢式PCR方法具有特异性强、敏感性高、重复性好等优点,可以准确快速的用于鸭圆环病毒的病原检测。     

Superinfection in ducks persistently infected with duck plague virus

Superinfections with homologous or heterologous strains of duck plague virus resulted in the deaths of birds persistently infected with duck plague virus. Not all birds that were superinfected died. Protection against mortality depended on the route of exposure, strain of the initial duck plague virus, and strain of the superinfecting virus.     

鸭圆环病毒LAMP可视化检测方法的建立

根据GenBank中DuCV基因序列,在保守区设计了6条特异性引物,并对反应条件进行优化,建立了一种适用于鸭圆环病毒（DuCV）的环介导等温扩增快速检测方法（LAMP）。该方法对H9亚型禽流感、小鹅瘟、鸭瘟、鸭肝炎、鸭副黏病毒均无扩增反应;且扩增反应只需在常规水浴锅中进行,1小时内即可完成反应;对DuCV模版DNA的最小检测限为10龟,灵敏度是一步法PCR的1000倍。本研究建立的LAMP方法简便、快速、灵敏、特异,适合在基层进行DuCV的快速检测。     

Outbreak of duck plague (duck herpesvirus enteritis) in numerous species of captive ducks and geese in temporal conjunction with enforced biosecurity (in-house keeping) due to the threat of avian influenza A virus of the subtype Asia H5N1

The continuing westward spread of avian influenza A virus of the subtype H5N1 in free-living and domestic birds forced the European Union and the German federal government to enhance all biosecurity measures including in-house keeping of all captive birds from October 20 to December 15, 2005. Movement of captive ducks and geese of many different species from a free-range system to tight enclosures and maintenance for prolonged times in such overcrowded sheds resulted in pronounced disturbance of natural behaviour, interruption of mating and breeding activities and possibly additional stress. Under these conditions the birds developed signs of severe disease and enhanced mortality twentyfour days later. A total of 17 out of 124 (14%) adult birds and 149 out of 184 year-old birds (81 %) died during the outbreak. A herpesvirus was isolated from many organs of succumbed ducks and geese that was identified as a duck plague herpesvirus by cross neutralization test using known antisera against duck plague virus. The published host range of duck plague comprises 34 species within the order Anseriformes. We report here on additional 14 species of this order that were found to be susceptible to duck plague virus. The exact source of the herpesvirus could not identified. However, low antibody titres in some ducks at day of vaccination indicate that at least some of the birds were latently infected with a duck plague herpesvirus. The remaining healthy appearing birds were subcutaneously vaccinated with a modified live duck plague vaccine (Intervet, Boxmeer, NL) that stopped losses and resulted in seroconversion in most of the vaccinated birds.     

番鸭小鹅瘟病直接荧光诊断试剂的研制

将抗番鸭GPV单抗腹水采用透析法标记异硫氰酸荧光素（FITC）,制备成抗GPV荧光抗体,研制检测GPV抗原的直接免疫荧光诊断方法。结果显示GPV荧光抗体仅与GPV阳性的组织切片或细胞呈现特异性荧光,与番鸭细小病毒（MPV）、番鸭呼肠孤病毒（MDRV）、鸭副粘病毒（DPMV）、鸭病毒性肝炎病毒（DHV）和正常番鸭组织切片不反应;与间接荧光方法的符合率为92．9％。表明GPV荧光抗体具有较好的特异性、敏感性和准确性,可用于临床快速诊断番鸭小鹅瘟病。     

番鸭细小病毒与鸭圆环病毒二重PCR方法的建立

根据基因库中鸭圆环病毒和番鸭细小病毒的基因序列,分别设计了两对特异性引物,通过对二重PCR扩增条件的优化,研究建立了可同时鉴别检测鸭圆环病毒和番鸭细小病毒的二重PCR方法。用该方法对同一样品中鸭圆环病毒和番鸭细小病毒的模板进行PCR扩增,结果均得到了与实验设计相符的351bp（鸭圆环病毒）和474bp（番鸭细小病毒）的扩增条带,而对鸭Ⅰ型肝炎病毒、鹅细小病毒、鸭副黏病毒、鸭瘟病毒和禽流感病毒等病原体的检测全为阴性。敏感性测定结果表明：该二重PCR技术最低能检出100fg的鸭圆环病毒和番鸭细小病毒DNA模板。研究建立的鸭圆环病毒和番鸭细小病毒的二重PCR方法,具有快速、敏感、特异、定量和重复性好等优点,可用于临床上鸭圆环病毒和番鸭细小病毒感染的检测。