表达H5亚型禽流感病毒HA基因的重组鸡痘病毒在水禽的免疫效力

利用表达 H5亚型禽流感病毒血凝素基因的重组鸡痘病毒 ( r FPV- HA)和油乳剂全病毒灭活疫苗分别接种商品鹅 ,评价疫苗在水禽的免疫保护作用。结果发现 ,免疫后 2 1 d,r FPV- HA免疫组 HI抗体检测为阴性 ,灭活疫苗免疫组HI抗体阳性率为 70 % ;用 H5亚型禽流感病毒攻击后 ,与阴性对照组相比 ,r FPV - HA免疫组鹅的口腔和泄殖腔排毒率降低 ,病理变化减轻 ,感染鹅易康复 ,保护率为 80 % ,总体保护效力达到灭活疫苗水平。结果表明 r FPV - HA免疫家鹅可诱导良好保护 ,显示出了良好的应用前景     

表达H9亚型禽流感病毒血凝素的重组HVT活疫苗免疫效力评价

为在SPF鸡和商品蛋鸡上评价一株表达H9亚型禽流感病毒(AIV)血凝素基因的重组HVT(r HVT-H9)活疫苗的免疫保护效力,试验将1日龄SPF鸡分为r HVT-H9疫苗组、灭活疫苗组和空白对照组,共3组(n=60,每组20只);1日龄商品蛋鸡除上述三组外另增加r HVT-H9疫苗与禽流感灭活疫苗(H9亚型,F株)联合免疫组,共4组(n=80,每组20只)。免疫后,通过血凝抑制试验检测血清中针对H9亚型AIV的抗体效价;并于免疫后28 d,将全部试验组及空白对照组以H9亚型AIV F株进行静脉注射攻毒。攻毒后第3、5天分别采集喉头、泄殖腔棉拭子检测排毒情况;并于攻毒后第10天将全部试验鸡进行剖检。结果显示:SPF鸡r HVT-H9疫苗组能达到100%免疫保护,而灭活疫苗组仅能达到80%保护;商品蛋鸡r HVT-H9疫苗组保护率为40%,灭活疫苗组保护率为70%,但联合免疫组能达到100%的免疫保护。因此,该重组病毒r HVT-H9疫苗可作为H9亚型禽流感的有效疫苗候选株,与现有商品化禽流感H9亚型灭活疫苗联合使用,效果更佳,为其防控提供一定的技术支持。     

Antibody Dynamics and Protective Efficacy of Recombinant Fowipox Virus Co-expressing Cytokine IL-6 Gene and HA Gene of H5 Subtype Avian Influenza Virus

为评价共表达鸡IL-6和H5亚型禽流感病毒HA基因重组鸡痘病毒(rFPV-AIH5 AIL6)的免疫抗体消长规律及免疫效力,将重组病毒通过颈部皮下注射和翅部皮下注射2种不同的免疫途径来免疫鸡群,结果发现,2种免疫途径中,重组鸡痘病毒对鸡体质量增加均无影响,而野生型鸡痘病毒均可抑制鸡体质量增加.翅部皮下注射疫苗组能够产生较高的血凝抑制(HI)抗体水平,免疫21 d后抗体水平达到高峰,28 d后开始下降,49 d时仍保持在一定的水平.免疫SPF鸡21 d后攻毒,表明该重组病毒能使经滴鼻攻毒的SPF鸡抵抗H5亚型AIV的致死性攻击,保护率为95％,与油苗组相同,与单表达H5亚型禽流感病毒HA基因重组鸡痘病毒组(40％)差异显著；攻毒后3、5、7d采集喉头、泄殖腔棉拭子检测排毒情况,结果发现第3天排毒率最高,其中rFPV-AIH5AIL6免疫组排毒率为最低,显示IL-6在rFPV-AIH5IL6免疫过程中起到了免疫佐剂的作用,这为研制新型的禽流感重组鸡瘟病毒疫苗奠定了基础.     

共表达鸡IL-6和H5亚型禽流感病毒HA基因重组鸡痘病毒免疫抗体消长规律及免疫效力研究

为评价共表达鸡IL-6和H5亚型禽流感病毒HA基因重组鸡痘病毒(rFPV-AIH5AIL6)的免疫抗体消长规律及免疫效力,将重组病毒通过颈部皮下注射和翅部皮下注射2种不同的免疫途径来免疫鸡群,结果发现,2种免疫途径中,重组鸡痘病毒对鸡体质量增加均无影响,而野生型鸡痘病毒均可抑制鸡体质量增加。翅部皮下注射疫苗组能够产生较高的血凝抑制(HI)抗体水平,免疫21d后抗体水平达到高峰,28d后开始下降,49d时仍保持在一定的水平。免疫SPF鸡21d后攻毒,表明该重组病毒能使经滴鼻攻毒的SPF鸡抵抗H5亚型AIV的致死性攻击,保护率为95%,与油苗组相同,与单表达H5亚型禽流感病毒HA基因重组鸡痘病毒组(40%)差异显著;攻毒后3、5、7d采集喉头、泄殖腔棉拭子检测排毒情况,结果发现第3天排毒率最高,其中rFPV-AIH5AIL6免疫组排毒率为最低,显示IL-6在rFPV-AIH5IL6免疫过程中起到了免疫佐剂的作用,这为研制新型的禽流感重组鸡痘病毒疫苗奠定了基础。     

禽白血病病毒对禽流感疫苗免疫效果的影响

为研究禽白血病病毒(ALV)对禽流感(AI)疫苗免疫效果的影响,本研究将1日龄SPF鸡经腹腔接种ALV HLJ09DH02株后,于21日龄免疫重组禽流感病毒(AIV)灭活疫苗(H5N1亚型,Re-8)或禽流感-新城疫重组二联活疫苗(r LH5-8),免疫后与未感染ALV的免疫对照鸡同时检测血清HI抗体并采用H5N1亚型高致病性AIV攻毒。结果显示:实验鸡感染ALV后HI抗体滴度均显著低于相应的未感染ALV的免疫对照组(p0.05)。感染ALV后,免疫Re-8灭活疫苗及其对照组攻毒后的排毒鸡数分别为4/10和0/10,存活率分别为90%和100%;感染ALV后,免疫r LH5-8活疫苗及其对照组攻毒后的排毒鸡数分别为10/10和0/10,存活率分别为0和100%;未感染ALV的AI免疫鸡均不发病、不死亡、不排毒;空白对照鸡攻毒后3 d内全部死亡并且排毒。本研究表明,ALV对AI灭活疫苗和活疫苗均具有显著免疫抑制作用,建议加强种鸡ALV的净化,降低对AI等疫苗免疫效果的影响。     

H7N9禽流感DNA疫苗的免疫保护效力研究

为检测H7N9禽流感病毒(AIV) DNA疫苗的免疫保护效力,本研究将H7N9禽流感疫苗株A/Chicken/Guangxi/SD098/2017 (H7N9)[CK/GX/SD098/17 (H7N9)]的HA基因密码子优化后,定向克隆至载体pCAGGS中构建重组质粒pCA-SD098。将pCA-SD098转染至293T细胞后,采用间接免疫荧光(IFA)和western blot检测,结果显示,HA蛋白可以在293T细胞中正确表达。分别利用15μg和20μg重组质粒pCA-SD098免疫3周龄SPF鸡,3周后以相同的剂量和方式加强免疫,加强免疫一周后通过鼻腔分别感染105EID50的同源高致病性AIV (HPAIV)CK/GX/SD098/17 (H7N9)和异源低致病性AIV (LPAIV) A/Chicken/Chongqing/SD057/2017 (H7N9)[CK/CQ/SD057/17 (H7N9)],攻毒10 d内记录免疫鸡发病和死亡情况,检测免疫鸡HI抗体水平,并于攻毒后第3 d、第5 d和第7 d无菌采集所有鸡的喉头和泄殖腔拭子,采用红细胞凝集试验(HA)检测病毒的滴度。结果显示,重组质粒pCA-SD098免疫后可以诱导SPF鸡产生较高水平的HI抗体,15μg pCA-SD098剂量免疫鸡后能够完全抵御H7N9 HPAIV的致死性攻击,20μg pCA-SD098剂量可以有效阻断H7N9 LPAIV的感染。攻毒后,所有免疫鸡无发病、无死亡、无排毒,本研究为质粒pCA-SD098作为防控H7N9禽流感的候选DNA疫苗提供了实验依据。     

H9N2亚型禽流感疫苗株的筛选及其免疫效果评价

通过交叉血凝试验,疫苗备用毒株免疫SPF鸡后再用同源和异源毒株攻毒,评价疫苗备用毒株对SPF鸡保护效果。结果显示,H9N2疫苗备用株免疫SPF鸡后,再用H9亚型流感病毒流行株攻毒,SPF鸡咽喉和泄殖腔排毒量大大降低。结果表明,本试验筛选的疫苗备用毒株对H9N2亚型流感病毒具有一定的保护率,完全符合疫苗株要求。     

H9N2亚型AIV不同攻毒方式排毒规律的研究

为了研究H9N2亚型禽流感目前的流行情况,从广东省某鸡场的发病鸡中分离到一株H9N2亚型禽流感病毒(A/Chicken/Guangdong/1104/2014),遗传进化分析显示该毒株属于BJ/94-like谱系h9.4.2.5分支,与经典疫苗株的HA基因相似性为89.3~90.7%,通过点眼滴鼻、静脉注射、肌肉注射的方式攻毒SPF鸡研究其攻毒后的排毒规律。结果表明:病毒通过点眼滴鼻和静脉注射方式攻毒能够很好地感染SPF鸡,并在攻毒后第3天排毒率达到100%,但是随后几天排毒率逐渐下降,点眼滴鼻组在攻毒后第7天排毒率下降为0;肌肉注射组则没能有效感染SPF鸡,攻毒后第3~9天的排毒率为16.7%~33.3%。说明静脉注射是较好的攻毒方式,能有效感染SPF鸡,SPF鸡攻毒后第3~5天的排毒率达到100%。     

Re-8株H5亚型禽流感灭活疫苗对野鸟源H5N8流感病毒的免疫保护性研究

为评估当前应用的Re-8株种毒相关禽流感灭活疫苗对野鸟源H5N8流感病毒的免疫预防效果,本研究将重组禽流感病毒(AIV)H5亚型Re-8株灭活疫苗(H5N1亚型,Re-8株)和重组AIV(H5+H7)灭活疫苗(H5N1Re-8株+H7N9 H7 Re-1株)分别以0.3 m L/只的剂量接种3周龄SPF鸡,免疫3周时进行HI抗体检测和攻毒试验。结果显示,上述2种疫苗免疫的SPF鸡血清中针对H5亚型Re-8株抗原的HI抗体均在8 log2以上,以105EID50的剂量、鼻腔感染途径攻击野鸟源H5N8病毒BHG/QH/2/16和BHG/Tibet/3/16后,所有免疫组鸡在14 d观察期内均全部健活,不排毒,而对照组鸡在攻毒后4 d内全部发病、死亡、排毒。现地免疫重组AIV H5亚型三价灭活疫苗(Re-6株+Re-7株+Re-8株)的商品蛋鸡直接进行HI抗体检测和攻毒试验,结果显示,免疫蛋鸡血清中针对Re-8株抗原的HI抗体平均滴度为8.3 log2,以相同攻毒方式和剂量分别攻击2株H5N8病毒后,免疫鸡也获得完全免疫保护,不发病、不死亡、不排毒。本研究结果表明,含有重组AIV H5 Re-8株抗原的灭活疫苗可有效防控野鸟源H5N8流感病毒,为我国及其他国家H5N8亚型禽流感免疫防控提供了科学依据。     

表达分泌型与膜结合型HH7N9 AIV血凝素蛋白重组NDV载体疫苗的免疫原性比较

研究以新城疫病毒(NDV)弱毒株LX为载体,构建了一株表达H7N9亚型禽流感病毒(H7N9 AIV)血凝素(HA)胞外区的重组病毒(LXs HA),并与一株前期构建的表达膜结合型HA的重组病毒(LXHAF)进行比较。生物学特性评价显示,两株病毒均为弱毒且在鸡胚中的繁殖性能较强。此外,LXs HA主要以分泌形式表达HA蛋白,而LXHAF则以膜结合形式表达HA。鸡的免疫试验显示,两株重组病毒在一次免疫后均能诱导针对NDV载体的血凝抑制抗体,但LXHAF能够诱导较高水平的H7N9 AIV特异性Ig Y抗体,而LXs HA不能诱导H7N9 AIV抗体应答。结果表明,表达膜结合型HA的重组病毒具有针对H7N9 AIV较好的免疫原性,而表达分泌型HA的重组病毒免疫原性较差。这为研发H7N9亚型禽流感载体疫苗提供新的信息与参考。     

Evaluation of the infectivity, length of infection, and immune response of a low-pathogenicity H7N2 avian influenza virus in specific-pathogen-free chickens

The H7N2 subtype of avian influenza virus (AIV) field isolate (H7N2/chicken/PA/3779-2/97), which caused the 1997-98 AIV outbreak in Pennsylvania, was evaluated for its infectivity, length of infection, and immune response in specific-pathogen-free (SPF) chickens. The composite findings of three clinical trials with various concentrations of virus indicated that this H7N2 subtype contained minimal pathogenicity for chickens. The concentration of the virus in the inoculum proved critical in the establishment of a productive infection in a chicken. Seven-day-old SPF chickens were not infected when inoculated with 10(0.7-2.0) mean embryo lethal dose (ELD50) of the H7N2 virus per bird. At this dose level, the immune response to this virus was not detected by the hemagglutination-inhibition (HI) test. Nonetheless, chickens at ages of 5 and 23 wk old tested were successfully infected when exposed to 10(4.7-5.7) ELD50 of H7N2 infectious doses per bird by various routes of administration and also by direct contact. Infected birds started shedding virus as early as 2 days postinoculation, and the period of virus shedding occurred mostly within 1 or 2 wk postinoculation (WPI). This H7N2 subtype of AIV induced a measurable immune response in all birds within 2 wk after virus exposure. Antibody titers were associated with AIV infectious doses and age of exposure of birds. Challenge of these infected birds with the same H7N2 virus at 5 and 10 WPI indicated the infective virus was recoverable from cloacal swabs at 3 days postchallenge and disappeared thereafter. In these challenged birds, the antibody levels as measured by the HI test spiked within 1-2 wk.     

新城疫病毒HI抗体效价与流行株感染排毒之间的相关性

为了分析免疫鸡群中新城疫强毒感染流行的原因,明确抗体效价与流行株感染排毒率之间的关系,本研究以LaSota为抗原制备新城疫灭活疫苗,并以0.02mL和0.4mL的量分别免疫3周龄的SPF鸡10只。免疫后7、14、21d分别测定免疫鸡血清中的抗体HI效价。免疫后21d以基因Ⅶd亚型新城疫流行株JS5/05进行攻毒,攻毒后每天观察试验鸡的临床症状,并于攻毒后3、5、7d采集试验鸡的喉气管与泄殖腔棉拭样品进行病毒分离,结果显示,免疫3周后0.02mL和0.4mL疫苗免疫组鸡的血清HI抗体平均效价分别为5.4log2和8.2log2;0.02mL免疫组在攻毒后的排毒率达到100%,且排毒时间较长,而0.4mL免疫组在攻毒后的排毒率明显降低,且排毒时间较短。上述结果表明新城疫抗体效价与流行株感染排毒率之间存在明显的负相关。     

H5N1亚型禽流感病毒感染海兰白鸡模型的建立

为建立H5N1亚型禽流感病毒感染海兰白鸡模型,本研究选取1株鹅源H5N1高致病性禽流感病毒A/goose/guangdong/1/96(H5N1)(简称GD1/96),测定其对4周龄海兰白鸡的半数致死量.感染模型试验中,将30只4周龄海兰白鸡随机分成3组,每组10只,5只直接感染,5只同居,试验组设置一个重复,将病毒液稀释至10^4.5EID₅₀,滴鼻、点眼各0.1 mL,对照组接种PBS,感染后24 h放入同居鸡;感染后连续观察14 d,记录死亡时间,每天采集咽喉拭子和泄殖腔拭子;感染组和同居组第3、5 天各剖解3只鸡,采集气管、肺脏、脑、脾脏、肾脏和十二指肠,进行病毒分离;qRT-PCR法分析感染组和同居组第3、5 天鸡肺组织中IFN-α和TNF-α的相对表达量.结果显示,GD1/96株的鸡胚半数感染量(EID₅₀)为10^-8.167/0.1 mL,对4周龄海兰白鸡的半数致死量为10^4.5 EID₅₀.感染模型试验结果显示,以10^4.5EID₅₀的攻毒剂量感染海兰白鸡,感染组鸡在感染后8 d全部死亡;在感染和同居3 d后,各组鸡的咽喉拭子和泄殖腔拭子均可检测到病毒;感染和同居后第3、5 天,各组鸡的6种组织中均可分离到高滴度的病毒;IFN-α和TNF-α在感染组和同居组的鸡肺脏组织中的表达量均显著增加(P <0.05).本试验建立了海兰白鸡的H5N1亚型禽流感病毒感染模型,为H5N1亚型禽流感病毒的致病机理及表达抗流感基因转基因鸡的研究奠定了基础.     

传染性喉气管炎新城疫鸡痘重组病毒免疫效力的研究

在表达鸡传染性喉气管炎病毒(ILTV)糖蛋白gB基因和新城疫病毒(NDV)F基因的重组鸡痘病毒(rF-PV-gB-F)安全性检验合格后,以5.0×101~5.0×104PFU不同含量按0.1mL/鸡的剂量免疫100只30日龄SPF鸡,30d后分组分别用ILTVWG株和NDVF48E9株强毒进行攻击。免疫鸡抗鸡痘病毒抗体都转为阳性,痘反应和接种剂量有关,重组疫苗的最小反应剂量为50PFU。重组疫苗可以诱发对新城疫和传染性喉气管炎的保护,0.1mL/鸡的接种量在500~5000PFU浓度范围内的免疫效果最好,对于ILTV攻击的发病保护率在70%以上,对NDV强毒攻击的抗死亡保护率可以达到80%,这为进一步考察疫苗的免疫效力试验以及进行田间试验奠定了基础。     

Immuno-PCR for one step detection of H5N1 avian influenza virus and Newcastle disease virus using magnetic gold particles as carriers

Detecting avian influenza virus (AIV) and Newcastle disease virus (NDV) at low concentrations from tracheal and cloacal swabs of avian influenza- and Newcastle disease-infected poultry was carried out using a highly sensitive immunological-polymerase chain reaction (immuno-PCR) method. Magnetic gold particles were pre-coated with a capture antibody, either a monoclonal anti-AIV/H5 or monoclonal anti-NDV/F and viruses serially diluted ten-fold from 10(2) to 10(-5)EID(50)/ml. A biotinylated detection antibody bound to the viral antigen was then linked via a streptavidin bridge to biotinylated reporter DNA. After extensive washing, reporter DNA was released by denaturation, transferred to PCR tubes, amplified, electrophoresed and visualized. An optimized immuno-PCR method was able to detect as little as 10(-4)EID(50)/ml AIV and NDV. To further evaluate the specificity and the clinical application of this IPCR assay for AIV H5N1 and NDV, the tracheal swab specimens, taken from chickens which were infected with H5N1/AIV, H9N2/AIV, H7N2/AIV, NDV, IBDV, IBV/H(120), were detected by IPCR. Our data demonstrated that this monoclonal antibody-based immuno-PCR method provides a platform capable of rapid screening of clinical samples for trace levels of AIV H5 and NDV in one step.     

Immunostimulant effect of a mixed herbal extract on infectious bursal disease virus (IBDV) vaccinated chickens in the context of a co-infection model of avian influenza virus H9N2 and IBDV

This study was conducted to assess the comparative effects of a mixed herbal extract (MHE) containing Ocimum sanctum, Withania somnifera, Emblica officinalis, Tinospora cordifolia, Mangifera indica, and Asphaltum (shilajit) on infectious bursal disease virus (IBDV)-vaccinated (VAC) chickens infected with IBDV and avian influenza virus (AIV) H9N2. The experiment included three groups (G1-G3): G1, the negative control group; G2, the VAC + challenged (Ch) group; and G3, the VAC + Ch + MHE group. MHE was orally administered continuously for 5 weeks post-vaccination (PV) with IBDV at 12 days of age, and the chicks were simultaneously challenged with virulent IBDV (intraocularly) and AIV H9N2 (intranasally) at 21 days PV. Blood and tissue samples as well as tracheal and cloacal swabs were gathered at different times PV and post-challenge. Immunological and haematological parameters, histopathological lesions, relative organ weights and final live weights revealed significant differences (P ≤ 0.05) between G2 and G3 groups. Furthermore, in the G3 group, the protection rates, ELISA and HI titers and CD4+/CD8+ ratio were significantly increased, whereas viral shedding titers and the heterophil/lymphocyte ratio were decreased. In conclusion, the oral administration of the mixed herbal extract for 5 weeks can stimulate the immune response to IBDV vaccination and relieves the pathogenicity of an AIV H9N2 and IBDV co-infection in chickens.     

新城疫LaSota活疫苗对基因Ⅶ型分离株的免疫保护性试验

从山东省发病鸡群分离鉴定了一株新城疫病毒(NDV),命名为SDLY01。经蚀斑纯化后进行毒力测定和序列分析表明分离株SDLY01属于基因Ⅶ型NDV强毒。20只7日龄SPF鸡免疫新城疫活疫苗LaSot a后14 d分别用NDV标准强毒F48E8和分离株SDLY01攻毒,同时设同日龄SPF鸡为对照组,未免疫任何疫苗。攻毒后观察10 d,免疫组在攻毒后食欲、精神均正常;对照组在攻毒后2～4d发病死亡,并表现ND典型的临床症状和病理变化。攻毒后第3、5、7、9 d对免疫组试验鸡取喉头、泄殖腔棉拭进行病毒分离,F48E8攻毒组病毒分离均为NDV阴性,SDLYO1攻毒组第5 d病毒分离NDV阳性,第3、7和9d病毒分离阴性。本研究结果表明LaSot a活疫苗对F48E8和SDLY01均能提供100%免疫保护,但不能完全抑制基因Ⅶ NDV分离株在体内的复制和排毒。     

Investigation of H7N2 avian influenza outbreaks in two broiler breeder flocks in Pennsylvania, 2001-02

An avian influenza (AI) outbreak occurred in meat-type chickens in central Pennsylvania from December 2001 to January 2002. Two broiler breeder flocks were initially infected almost simultaneously in early December. Avian influenza virus (AIV), H7N2 subtype, was isolated from the two premises in our laboratory. The H7N2 isolates were characterized as a low pathogenic strain at the National Veterinary Services Laboratories based on molecular sequencing of the virus hemagglutinin cleavage site and virus challenge studies in specific-pathogen-free leghorn chickens. However, clinical observations and pathologic findings indicated that this H7N2 virus appeared to be significantly pathogenic in meat-type chickens under field conditions. Follow-up investigation indicated that this H7N2 virus spread rapidly within each flock. Within 7 days of the recognized start of the outbreak, over 90% seroconversion was observed in the birds by the hemagglutination inhibition test. A diagnosis of AI was made within 24 hr of bird submission during this outbreak using a combination of virus detection by a same-day dot-enzyme-linked immunosorbent assay and virus isolation in embryonating chicken eggs. Follow-up investigation revealed that heavy virus shedding (90%-100% of birds shedding AIV) occurred between 4 and 7 days after disease onset, and a few birds (15%) continued to shed virus at 13 days post-disease onset, as detected by virus isolation on tracheal and cloacal swabs. AIV was not detected in or on eggs laid by the breeders during the testing phase of the outbreak. The two flocks were depopulated at 14 days after disease onset, and AIV was not detected on the two premises 23 days after depopulation.