酵母诱导表达鸡IFN-α抗IBDV效果及其对信号分子PI3K和NF-κB的影响

试验旨在研究酵母表达鸡IFN-α抗传染性法氏囊病病毒(IBDV)的效果及对其淋巴细胞信号分子PI3K和NF-κB p65的影响。将40只10日龄非免疫雏鸡随机分为IFN-α组和空白对照组,持续注射给药3d后,于第3次给药后第1天开始每天心脏采血,持续3d,并于最后一次采血后对雏鸡进行攻毒,在攻毒后开始每天心脏采血,连续3d。通过MTT法检测淋巴增殖活性情况,ELISA测定不同时间段淋巴细胞中PI3K的水平、细胞中总NF-κB p65、细胞核中NF-κB p65蛋白表达含量及IBDV抗原量。结果显示,与对照组相比,攻毒前,IFN-α对淋巴增殖活性、细胞中PI3K和NF-κB p65的表达具有极显著地促进作用(P0.01);与攻毒前相比,攻毒后第1和2天IFN-α组淋巴增殖活性、细胞中PI3K和NF-κB p65的表达极显著增加(P0.01),IFN-α对IBDV-Ag具有极显著地抑制效果(P0.01)。这些结果表明,IFN-α可通过增加PI3K激活NF-κB信号通路以增强机体免疫反应,并对IBDVAg有显著的抑制作用,本试验为IFN-α免疫增强和抗病毒作用的研究提供科学依据。     

紫锥菊、黄芪对法氏囊病雏鸡的肝损伤及脾脏NK细胞活性的影响

给14日龄雏鸡灌服不同浓度紫锥菊、黄芪合剂,连续灌服7d,21日龄时雏鸡接种传染性法氏囊病病毒(IB-DV),来观察雏鸡血液转氨酶活性及脾脏NK细胞活性的变化。结果显示,攻毒后4d和攻毒后11d时用药组与攻毒对照组相比,谷草转氨酶(Ast)、谷丙转氨酶(Alt)均有所降低,差异显著(P<0.05);攻毒后11d时Ast/Alt比值极显著低于攻毒对照组(P<0.01)。脾脏NK细胞活性在感染后14d,高剂量组活性极显著高于攻毒对照组和空白对照组(P<0.01),感染21d后各组差异不显著。结果表明,紫锥菊、黄芪合剂在雏鸡感染IBDV后12d前后一段时间可显著降低传染性法氏囊病病毒对肝脏造成的损伤,促进脾脏NK细胞的活性,增强机体的防御功能,有利于该疾病的防治。     

雏鸡感染传染性法氏囊病病毒后红细胞免疫功能的观察

用经传染性法氏囊病(IBD)弱毒苗免疫和未免疫的蛋鸡为试验动物,检测了感染IBDV后雏鸡的红细胞C3b受体(RBC-C3bR)花环率和红细胞免疫复合物(RBC-IC)花环率。试验结果表明,雏鸡在感染IBDV后红细胞C3bR花环率显著(P<0.01或P<0.05)低于B组,红细胞免疫复合物(IC)花环率极显著(P<0.01)高于B组,说明感染IBDV后雏鸡红细胞的免疫调节功能受到明显抑制。     

重组鸡白细胞介素-6(rChIL-6)对传染性法氏囊灭活疫苗的免疫增强机理研究

为了评价重组鸡白细胞介素-6(rChIL-6)蛋白在鸡体内的免疫增强作用机理,本研究将40只7日龄健康SPF鸡随机平均分为4组以进行rChIL-6蛋白对鸡传染性法氏囊病毒(IBDV)灭活疫苗免疫增强作用试验,于14日龄时进行免疫接种和rChIL-6蛋白注射。分别检测4组鸡免疫前和免疫后不同时间血清中IBDV抗体水平、不同细胞因子表达水平、脾淋巴细胞增殖活性和外周血T淋巴细胞增殖活性等指标以揭示rChIL-6蛋白在机体内对疫苗的免疫增强机理。结果表明:免疫后7⁴9d,rChIL-6蛋白与IBDV灭活疫苗共免疫组(第4组)鸡血清中IBDV抗体水平极显著高于IBDV灭活疫苗对照组(P<0.01);免疫后749d,rChIL-6蛋白与IBDV灭活疫苗共免疫组(第4组)鸡血清中IBDV抗体水平极显著高于IBDV灭活疫苗对照组(P<0.01);免疫后7³5d,第4组鸡血清中的白细胞介素-4、6、2(ChIL-4、ChIL-6、ChIL-2)和γ干扰素(ChIFN-γ)等4种细胞因子的表达水平极显著高于其他3组对照组(P<0.01),α干扰素(ChIFN-α)的表达水平于免疫后1435d,第4组鸡血清中的白细胞介素-4、6、2(ChIL-4、ChIL-6、ChIL-2)和γ干扰素(ChIFN-γ)等4种细胞因子的表达水平极显著高于其他3组对照组(P<0.01),α干扰素(ChIFN-α)的表达水平于免疫后14³5d期间极显著高于其他3组对照鸡(P<0.01);rChIL-6蛋白单独应用后可显著提高机体的ChIL-4、ChIL-6和ChIL-2的表达水平,但对ChIFN-γ、ChIFN-α的表达水平无明显影响。免疫后735d期间极显著高于其他3组对照鸡(P<0.01);rChIL-6蛋白单独应用后可显著提高机体的ChIL-4、ChIL-6和ChIL-2的表达水平,但对ChIFN-γ、ChIFN-α的表达水平无明显影响。免疫后7¹4d,第4组鸡的脾淋巴细胞增殖活性显著高于其他3组对照鸡(P<0.01);免疫后714d,第4组鸡的脾淋巴细胞增殖活性显著高于其他3组对照鸡(P<0.01);免疫后7²1d,第4组鸡的外周血T淋巴细胞的增殖活性稍高于其他3组对照组(P>0.05)。结果表明,rChIL-6在体液免疫和细胞免疫水平对鸡传染性法氏囊灭活疫苗免疫后均具有免疫增强作用;对机体体液免疫应答的增强能力高于细胞免疫应答。     

中药对雏鸡感染IBDV前后法氏囊相对重量及血清γ—球蛋白动态变化的影响

在雏鸡日粮中添加1%的中药,于30日龄时进行人工感染 IBDV 强毒,其结果添加中药组攻毒后3天法氏囊相对重量增加不明显(P>0.05),而未添加中药组却明显增加(P<0.05)。攻毒后10天时中药纽法氏囊的相对重量接近正常(P>0.05)。此外,攻毒后3～10天中药组血清γ-球蛋白的百分含量显著提高(P<0.05),且攻毒后10天与攻毒前相比差异极显著(P<0.01)。     

传染性法氏囊病毒感染SPF雏鸡哈德尔腺和盲肠扁桃体TGF-β1 mRNA表达变化

为探索鸡传染性法氏囊病病毒(IBDV)感染对SPF雏鸡消化道和呼吸道局部黏膜免疫组织转化生长因子-β1 (TGF-β1)mRNA表达的影响,以21日龄SPF雏鸡为研究对象,应用荧光定量PCR方法,对SPF雏鸡感染IBDV后,其哈德尔腺和盲肠扁桃体中TGF-β1 mRNA表达的动态变化进行检测.结果发现,21日龄SPF雏鸡感染IBDV强毒后1～28 d,其上述局部黏膜免疫组织中TGF-β1 mRNA表达均不同程度的高于对照雏鸡,其中哈德尔腺TGF-β1 mRNA表达于病毒感染后3～28 d极显著高于对照雏鸡(P＜0.01);而盲肠扁桃体的上述被检指标于病毒感染后3～14 d极显著高于对照雏鸡(P＜0.01).表明IBDV感染所致的雏鸡免疫抑制与雏鸡消化道和呼吸道局部黏膜免疫组织TGF-β1 mRNA表达增加密切相关.本研究为进一步阐明IBDV的免疫致病机制提供了重要的参考依据.     

黄芪多糖对人工感染IBDV雏鸡红细胞免疫功能的影响

将由未免疫接种腔上囊病疫苗鸡的种蛋孵化而来的160只1日龄海兰白雏鸡随机分为A、B、C和D共4组。A组为对照组，B、C和D组于26日龄时按每只0．3mL的剂量用IBDV攻毒。A、B组未用黄芪多糖（APS）处理，C、D组从攻毒当日起连续胸肌注射APS6d，剂量分别为每只鸡5mg和10mg。分别在21、29、32、35、38日龄时心脏采血1～3mL，测定E—C3bRR、E—ICRR、ERER和ERIR。结果显示，雏鸡感染IBDV后可使E-C3bRR和ERER显著降低（P〈0．01）；APS处理组E-C3bRR、E-ICRR、ERER均高于A、B组（P〈0．01），其中D组最高，而ERIR则与A组相似。证实，雏鸡感染IBDV后红细胞免疫功能低下，而APS可显著提高其红细胞免疫功能。     

SPF雏鸡感染REV后免疫器官免疫功能的动态变化

试验通过对 1日龄 SPF雏鸡人工感染网状内皮组织增殖病病毒 ( REV) ,观察了免疫器官胸腺、法氏囊和脾脏 T、B淋巴细胞增殖反应的动态变化。结果表明 ,雏鸡感染 REV后胸腺 T淋巴细胞的增殖反应于7、2 1、4 2和 4 9日龄明显降低 ( P<0 .0 5) ,于感染后 1 4、2 8和 3 5日龄极显著减弱 ( P<0 .0 1 ) ;脾脏 T淋巴细胞的增殖反应分别于感染后 1 4～ 2 8d和 3 5～ 4 9d较对照组雏鸡明显或极明显降低 ( P<0 .0 5,P<0 .0 1 )。法氏囊和脾脏 B淋巴细胞增殖反应分别在感染后 7～ 4 9d和 1 4～ 4 9d极显著减弱 ( P<0 .0 1 )。免疫器官胸腺重量和脾脏重量与体重比值及体重分别于感染后 7～ 4 9、7～ 3 5、7～ 4 9d明显降低 ( P<0 .0 5,P<0 .0 1 )。说明SPF雏鸡感染 REV后 ,免疫器官 (胸腺、法氏囊和脾脏 )淋巴细胞发生变性坏死、数量减少和功能降低 ,机体的细胞免疫和体液免疫功能均发生明显降低或抑制     

表达牛乳铁蛋白肽的重组鸡源乳酸杆菌抗IBDV感染的研究

为探究表达牛乳铁蛋白肽的重组鸡源乳酸杆菌pPG-XLFEC/M11对雏鸡抵抗传染性法氏囊病病毒(IBDV)感染的作用,本研究利用IBDVCEF94株感染DF-1细胞,通过CCK-8法检测病毒感染后的细胞活性,利用qRT-PCR检测病毒载量,结果显示重组乳酸菌表达的牛乳铁蛋白肽可以抑制IBDV在DF-1细胞内的增殖;以重组乳酸菌pPG-XLFEC/M11饲喂雏鸡,利用IBDV强毒UK661株进行攻毒实验,结果显示饲喂重组菌的雏鸡相比于对照组,IBDV的感染对其组织器官的损伤较小,总免疫球蛋白IgG和SIgA含量增加,细胞因子IL-6、IL-8、TNF-琢和IFN-酌以及TLR2的mRNA转录水平均显著升高,且提高了雏鸡的存活率。本实验结果表明,重组鸡源乳酸杆菌表达的牛乳铁蛋白肽可以增强机体的免疫应答水平,对雏鸡具有一定的抗IBDV感染的作用。本研究为表达牛乳铁蛋白肽的重组鸡源乳酸杆菌在生产中的应用以及为禽类疾病的预防奠定基础。     

鸡Akirin2和GM-CSF促进传染性法氏囊病病毒VP2基因DNA疫苗免疫效果的研究

为提高鸡传染性法氏囊病病毒VP2基因DNA疫苗的免疫效力,将IBDV VP2、鸡Akirin2和鸡GM-CSF三基因编码区串联后克隆到真核表达载体pTriEx-4上,用构建的pT-VP2-Akirin2-GM-CSF重组质粒体外转染293T细胞,然后提取表达产物进行SDS-PAGE分析。结果在58、35、22ku附近各有一蛋白条带。用pT-VP2-Akirin2-GM-CSF免疫SPF鸡,加强免疫2周后,攻IBDV BC6-85强毒,4d后统计保护率。结果发现,注射14d后,pT-VP2-Akirin2-GM-CSF质粒免疫组血清中能检测到特异性IBDV抗体和IBDV中和抗体,第28天,该组鸡的抗体滴度极显著高于空白对照组(P0.01),该组鸡外周血淋巴细胞增殖能力也极显著高于空白对照组(P0.01)。说明VP2-Akirin2-GM-CSF三基因串联DNA疫苗有很好的应用前景。     

Effects of Compound Chinese Herbal Medicinal Polysaccharide Extraction on NF-κB,TNF-α and IL-6 mRNA Expression in Different MHC B-Lβ Ⅱ Genotype of Chickens Lymphocyte

In order to explore the effect of compound Chinese herbal medicines polysaccharides(cCHMPS) on immunomodulatory in different MHC B-Lβ Ⅱ genotype chickens,200 White feather broiler were chosen and PCR-SSCP technique was applied to analyze the polymorphism of MHC B-Lβ Ⅱ gene. The peripheral blood were collected according to the different MHC B-Lβ Ⅱ genotype,and the lymphocytes were isolated from peripheral blood and the cCHMPS were added with a final concentration of 100,75,50 and 0 μg/mL for co-culturing 24 h.Then the expression of NF-κB,TNF-α,IL-6 mRNA in lymphocyte using Real-time PCR method were detected. The results showed that:Compared with the control group,different does of cCHMPS could significantly improve NF-κB,TNF-α,IL-6 mRNA expression levels in chicken with different MHC B-Lβ Ⅱ genotypes (P<0.05), and when the cCHMPS concentration was 50 μg/mL,the NF-κB,TNF-α,IL-6 mRNA expression levels in lymphocyte of AB and AA genotype chicken were significantly higher than that of other groups (P<0.05).The NF-κB,IL-6 mRNA expression levels of AC genotype chicken were significantly higher than the other groups (P<0.05). The TNF-α mRNA expression levels of AC genotype chicken were significantly higher than the other groups when cCHMPS was 100 μg/mL (P<0.05).There results indicated that the cCHMPS could stimulate NF-κB,TNF-α,IL-6 mRNA expression in different MHC B-Lβ Ⅱ genotype chickens,and the optimum immunomodulatory does were different in each MHC B-Lβ Ⅱ genotype chicken.     

绿原酸对FAdV-4的抑制作用及其对鸡胚肝脏炎性因子和信号分子表达的影响

试验旨在研究绿原酸对禽腺病毒4型(FAdV-4)的抑制作用及其对鸡胚肝脏炎性因子和信号分子表达的影响。将9日龄SPF鸡胚随机分为7组:对照组、5个不同浓度的绿原酸组和FAdV-4感染组。绿原酸组经尿囊腔注射不同浓度的绿原酸,48 h后与FAdV-4感染组均经尿囊腔接种0.2 mL ELD₅₀为1.36×10⁷拷贝的FAdV-4病毒,对照组注射等体积的生理盐水。用MTT法检测绿原酸的安全浓度(IC₅₀),计算每枚鸡胚接种绿原酸的含量;接种病毒后72 h后,剖检并无菌收集鸡胚肝脏进行病理观察,实时荧光定量PCR法检测病毒载量,ELISA法测定肝脏细胞因子白介素-1β(IL-1β)、IL-6和肿瘤坏死因子-α(TNF-α)及信号分子磷脂酰肌醇-3-激酶(PI3K)、核转录因子-κB(NF-κB)和NF-κBp65含量。MTT法测定结果表明,绿原酸对鸡胚成纤维细胞的IC₅₀为28.6 μg/mL,每枚鸡胚接种0.2 mL的绿原酸含量分别为858、429、214.5(≈215)、107.25(≈107)和53.125(≈53) μg。FAdV-4病毒接种72 h后,FAdV-4感染组鸡胚肝脏边缘钝圆、肿大,变脆,呈土黄色或黄色,甚至出现坏死;随着绿原酸浓度增加,绿原酸组鸡胚肝脏肿胀渐不明显,坏死灶和出血瘀点减少;肝脏内病毒明显增殖,载量为7.8 log₂拷贝/mg,107 μg绿原酸即可显著抑制FAdV-4在鸡胚肝脏中的增殖(P<0.05)。炎性细胞因子检测结果表明,与对照组相比,FAdV-4可极显著增加鸡胚肝脏中IL-1β、IL-6和TNF-α的含量(P<0.01);53 μg绿原酸组IL-1β和TNF-α含量极显著增加(P<0.01),107 μg绿原酸组IL-6和TNF-α的含量均显著增加(P<0.05)。与FAdV-4感染组相比,107 μg绿原酸组IL-1β和TNF-α的含量均极显著降低(P<0.01),215和429 μg绿原酸组IL-6的含量显著降低(P<0.05),858 μg绿原酸组IL-6的含量极显著降低(P<0.01)。细胞信号分子检测结果表明,与对照组相比,FAdV-4可极显著增加PI3K、NF-κB和NF-κBp65的含量(P<0.01);53 μg绿原酸组PI3K、NF-κB和NF-κBp65的含量均显著增加(P<0.05)。与FAdV-4感染组相比,53 μg绿原酸PI3K和NF-κBp65的含量均显著降低(P<0.05);215 μg绿原酸组NF-κB的含量显著降低(P<0.05),NF-κBp65的含量极显著降低(P<0.01);当绿原酸达429 μg时,PI3K、NF-κB和NF-κBp65的含量均极显著降低(P<0.01)。本试验结果表明,绿原酸能够抑制FAdV-4的增殖,且剂量依赖性地抑制炎性因子和信号分子的表达,说明绿原酸可用于鸡抗病毒和炎性相关疾病的防控。     

Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-�� on protective immunity by a DNA vaccine against IBDV in chickens

This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-γ (ChIFN-γ) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-ã was constructed. Twice at 2-week intervals, two-week-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-γ were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-γ was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-γ groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-γ had no significant effect.     

腹水综合征肉鸡空肠黏膜组织结构及相关基因表达分析

试验旨在研究肉鸡腹水综合征发生发展过程中空肠核因子κB （NF-κB）及紧密连接蛋白（闭合蛋白（occludin）、紧密连接蛋白-1（claudin-1）和胞质紧密黏连蛋白1（zonula occluden 1,ZO-1）） mRNA相对表达量,以及绒毛长度、隐窝深度和绒隐比的变化,为肉鸡腹水综合征的防治提供理论依据。选取40羽1日龄罗斯肉鸡常规饲养7 d后,随机分为对照组和模型组（饲料添加3%猪油和4%鱼粉,饮水添加0.12% NaCl,9~11℃低温饲养）,35日龄检测体重,并取心脏组织测腹水心脏指数,取空肠组织以HE染色切片测量绒毛长度和隐窝深度,实时荧光定量PCR检测空肠NF-κB和紧密连接蛋白的基因表达量。结果表明,与对照组相比,模型组肉鸡体重极显著下降（P<0.01）,腹水心脏指数极显著升高（P<0.01）,空肠绒毛长度、绒隐比极显著下降（P<0.01）,隐窝深度极显著升高（P<0.01）,NF-κB mRNA相对表达量极显著升高（P<0.01）,occludin、claudin-1和ZO-1 mRNA相对表达量极显著下降（P<0.01）。表明肠道黏膜结构与功能异常及肠通透性增高促进了肉鸡腹水综合征的发生发展。     

相同营养条件下五种鸡肉肌苷酸及其相关代谢物含量的比较研究

选用健康正常的1日龄宫廷黄鸡、快大黄鸡、广西黄鸡、罗曼蛋鸡和白羽肉鸡各120只,在相同营养条件下饲养至98日龄时屠宰,测定并比较各品种鸡的肌肉肌苷酸及其相关代谢物含量。试验结果如下,对于HYP的含量,罗曼蛋鸡极显著高于宫廷黄鸡和快大黄鸡(P<0.01),显著高于广西黄鸡(P<0.05),其它各品种间差异不显著(P>0.05)。对于IMP含量,宫廷黄鸡和快大黄鸡极显著高于白羽肉鸡(P<0.01)并显著高于罗曼蛋鸡(P<0.05),白羽肉鸡显著低于广西黄鸡(P<0.05),其它各品种间差异不显著(P>0.05)。对于INO含量,宫廷黄鸡和广西黄鸡极显著低于白羽肉鸡(P<0.01)。对于AMP含量,白羽肉鸡极显著高于广西黄鸡、宫廷黄鸡和快大黄鸡(P<0.01),罗曼蛋鸡显著高于广西黄鸡和快大黄鸡(P<0.05),宫廷黄鸡极显著低于罗曼蛋鸡(P<0.01)。对于ADP含量,宫廷黄鸡、广西黄鸡和快大黄鸡显著高于罗曼蛋鸡和白羽肉鸡(P<0.05)。校正IMP含量在各品种间均无显著差异(P>0.05)。     

坝上长尾鸡与海兰褐鸡肌肉营养成分对比研究

坝上长尾鸡是河北省唯一被列入《中国畜禽遗传资源志》的地方家禽品种。为了更好地保护和开发利用坝上长尾鸡资源,本试验选取相同饲养管理条件下200日龄的该鸡种和海兰褐鸡各180只,每组随机分为5个重复,每个重复36只。在同一条件下常规饲养,于230日龄每组挑选10只鸡屠宰,对肌肉中主要营养成分进行了比较分析。结果显示:1)坝上长尾鸡肌肉中的粗蛋白质和粗脂肪含量与海兰褐鸡相比差异不显著(P0.05)。2)2个鸡种间胸肌、腿肌中的必需氨基酸含量差异不显著(P0.05);坝上长尾鸡肌肉中的呈味氨基酸(谷氨酸、天冬氨酸、甘氨酸和丙氨酸)含量显著高于海兰褐鸡(P0.05),腿肌中的苦味氨基酸——苯丙氨酸含量显著低于海兰褐鸡(P0.05)。3)坝上长尾鸡腿肌中的不饱和脂肪酸、饱和脂肪酸和总脂肪酸含量显著低于海兰褐鸡(P0.05);坝上长尾鸡腿肌中的花生四烯酸和二十二碳六烯酸含量显著高于海兰褐鸡(P0.05)。4)坝上长尾鸡胸肌中的镁(P0.01)和铁(P0.05)含量显著或极显著高于海兰褐鸡,腿肌中的钠(P0.05)、镁(P0.01)和铁(P0.05)的含量显著或极显著高于海兰褐鸡。由此可见,坝上长尾鸡和海兰褐鸡的肌肉均品质优良,且坝上长尾鸡肌肉品质优于海兰褐鸡。     

感染IBDV雏鸡血液激素水平和ANAE阳性淋巴细胞动态变化

为探讨内分泌活动及细胞免疫反应在鸡抗传染性法氏囊病毒（ＩＢＤＶ）感染中的调节作用机制而进行了本研究。结果表明,攻毒后１～５ｄ内,未免疫攻毒鸡（Ａ组）、免疫攻毒鸡（Ｂ组）血浆皮质酮均明显上升,而未免疫未攻毒鸡（Ｃ组）则否。Ａ、Ｂ、Ｃ３组血浆Ｔ４水平攻毒前后无明显变化,Ｔ３除在攻毒后第３天Ａ组明显高于Ｂ、Ｃ组外,其他时间无明显差异性变化。血液ＡＮＡＥ阳性淋巴细胞（％）在攻毒后的１～５ｄ内,Ａ组和Ｂ组明显下降,以后回升,至２８ｄ回复到攻毒前水平,并接近于Ｃ组。Ａ组的ＡＮＡＥ阳性淋巴细胞（％）与皮质酮呈显著负相关,与Ｔ３和Ｔ４无明显相关性。     

Reduction of infectious bursal disease virus replication by shRNAs targeting the VP1 and VP2 genes driven by chicken U6 promoter

Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease in young chickens and results in considerable economic losses for the poultry industry. To suppress the replication of IBDV, two short hairpin RNAs (shRNAs) were designed for targeting the VP1 and VP2 genes of IBDV. Recombinant plasmids carrying each shRNA or two shRNAs were constructed based on vector pSilencer2.1-U6 in which the human U6 promoter was replaced with chicken U6 promoter. In chicken embryo fibroblasts, transfection with these shRNA plasmids 24 h before infection with IBDV B87 reduced 50% tissue culture infectious doses (TCID₅₀) from 10^8.75 TCID₅₀/0.1 mL to 10^3.75–10^1.0 TCID₅₀/0.1 mL. In 10-day old specific pathogen-free (SPF) chicken embryos, incubation with a mixture of IBDV B87 and a shRNA plasmid via the allantoic cavity resulted in 100% mortality and high IBDV virus titer in the control group but 25–0% mortality and near normal embryo development in the specific shRNA groups; additionally, IBDV VP1 and VP2 mRNA levels were reduced by 72–95% in the shRNA groups as compared with the control groups. When challenged with a virulent strain IBDV GX8/99, 14-day-old chickens pre-treated with the single shRNA plasmids or the dual shRNA plasmid showed approximately 70% or 90% survival at 5 days post-challenge while those pre-treated with control plasmid or saline had less than 5% survival. The current study suggests that two IBDV shRNAs expressed by a plasmid under chicken U6 promoter could effectively and synergistically reduce IBDV replication in vitro and in vivo.     

Protective immune responses of recombinant VP2 subunit antigen of infectious bursal disease virus in chickens

Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease and poses a huge threat to poultry industry. The risks associated with conventional attenuated viral vaccines make it indispensable to probe into the development of novel and rationally designed subunit vaccines which are safer as well as effective. VP2 is the major host-protective antigen found in IBDV capsid. It encompasses different independent epitopes responsible for the induction of neutralizing antibody. Here, we report the efficacy of the immunodominant fragment of VP2 which induces both humoral and cellular immunity against infectious bursal disease. A 366bp fragment (52-417bp) of the VP2 gene from an IBDV field isolate was amplified and expressed in Escherichia coli as a 21kDa recombinant protein. The efficacy of rVP2(52-417) antigen was compared with two commercial IBDV whole virus vaccine strains. The rVP2(52-417) induced significantly high antibody titres in chicken compared to commercial vaccines and the anti-rVP2(52-417) sera showed reactivity with viral antigens from both commercial strains (P<0.0001) and field isolates. Also, the chicken splenocytes from rVP2(52-417) immunized group showed a significantly high proliferation (P<0.01) compared to other groups, which implies that the rVP2(52-417) fragment contains immunogenic epitopes capable of eliciting both B and T cell responses. Further, rVP2(52-417) conferred 100% protection against vIBDV challenge in the immunized chickens which was significantly higher (P<0.001) compared to 55-60% protection by commercial vaccine strains. Hence, the study confirms the efficacy of the immunodominant VP2 fragment that could be used as a potent vaccine against IBDV infection in chicken.     

Study on Efficacy of Baihu Dingchuan Oral Liquid

In order to verify the effect of Baihu Dingchuan oral liquid of new veterinary drugs,the effects of infectious bronchitis virus (IBV) on the proliferation inhibition test, tracheal cilia movement test and SPF chickens infected with IBV were tested.The test solution was mixed with an equal amount of IBV solution,inoculation of 11-day-old SPF chicken embryo,incubation at 37℃ for 168 h.RT-PCR was performed to detect IBV in kidney of live chick embryo.The results showed that the survival rate of 168 h chicken embryo was 80% in the drug group,but the positive control group was 20%, and there was significant difference between two groups (P<0.05). In the test drug group, no band was detected by RT-PCR, but the target band appeared in the positive control group.After 40 days old SPF chickens were administered continuously for 3 days, 0.02 mL of ink was injected into the trachea. The distance of ink movement in 1 min was measured. The results showed that the migration distance of ink was extremely significantly higher than that of saline control group (P<0.01).In the 17-day-old SPF chickens, IBV-M41 was infused into the nasal mucosa with 0.3 mL. After 24 h, 1 mL/kg was administered for 5 days. The results showed that there were significant differences in clinical symptom score and cure rate between the test drug group and the control group (P<0.05). There was no significant pathological changes in the tracheal mucosa of the experimental group, but the pathological changes in the positive control group were significant. The above results showed that Baihu Dingchuan oral liquid had good inhibitory effect on IBV in vitro, which could promote the movement of tracheal cilia, and had good clinical control effect on infectious bronchitis.