鸭致病性大肠埃希菌的分离鉴定与毒力基因检测及菌蜕制备

从江苏省某水禽场的疑似感染致病性大肠埃希菌的病鸭中分离获得鸭致病性大肠埃希菌野毒株;通过玻板凝集和试管凝集试验确定其血清型,采用PCR进行分群分析和毒力基因检测;扩增φX_(174)噬菌体裂解蛋白E的基因序列,构建温控型表达质粒pBV221–E,并将其电击转化入分离毒株,升温诱导E蛋白表达制备菌蜕;通过测量菌液A600 nm值和在透射电镜下观察细菌形态,评估菌蜕构建效果;用该菌蜕进行灭活处理和无菌检测后,对雏鸭进行免疫,用间接ELISA法检测血清IgG水平。结果表明:该大肠埃希菌的血清型为O24,属于B1群,具有毒力基因fimC、csgA和iroN;获得的鸭致病性大肠埃希菌菌蜕裂解率为99.96%;经透射电镜观察发现,制备获得的菌蜕表面有明显孔道,细胞质从孔道溢出,细胞膜皱缩变形;抗体水平检测结果显示,二免后菌蜕免疫组雏鸭血清IgG水平显著提高。可见,通过将pBV221–E重组质粒转化至鸭致病性大肠埃希菌分离株,调节细菌培养温度诱导E蛋白表达,能制备鸭致病性大肠埃希菌B1群O24型野毒株菌蜕。该菌蜕可诱发机体产生体液免疫。     

Effect of a Chinese herbal formula Astragalus immunomodulator on immune function of chickens

In order to investigate the immunomodulatory effects and the mechanism of a Chinese herbal medicine, Zengmiansan (Astragalus immunomodulator), on immune function of chickens, three hundred 1-day-old chickens were assigned randomly into 5 groups, i.e., the blank control group, the Chinese herbal medicine Zengmiansan (ZMS)-treated group, the cyclophosphamide group, the cyclophosphamide plus levamisole group, the cyclophosphamide plus ZMS group and the control group. All chickens were immunized with Lasota vaccine by nosedrip or eye-drop at the age of 6 days. Newcastle Disease (ND) antibody titers, growth indexes of the spleen, thymus and bursa of Fabricius, the concentrations of CD₄ ⁺ lymphocytes and CD₈ ⁺ lymphocytes in spleen, thymus and peripheral blood, and the apoptosis of splenocytes, thymocytes and bursa of Fabricius cells were observed at the ages of 14, 21, 28 and 35 days, respectively. Our results indicated that the NDV antibody titers of chickens in the Chinese herbal medicine ZMS-treated group at the ages of 14 and 21 days were significantly higher than that of the other groups (P<0.01). The growth indexes of immune organs, the concentrations of CD₄ ⁺ lymphocytes and the ratio of CD₄ ⁺/CD₈ ⁺ T lymphocytes of chickens in the Chinese herbal medicine ZMS- treated group at the ages of 14, 21 and 28 d were significantly higher than those of the other groups (P<0.01). The apoptotic splenocytes, thymocytes and bursa of Fabricius cells of chickens in the ZMS-treated group were significantly lower than the other groups (P<0.01) at the ages of 14, 21 and 28 days.     

The SNPs C.513A>T in the MHC B-F gene and rs15001532 in the SPOCK1 gene are associated with Salmonella pullorum disease resistance in chickens

This study was designed to identify molecular markers single nucieotide polymorphisms(SNPs) of MHC B-F gene and SPOCK1) associated with Salmonella pullorum disease susceptibility/resistance.A two-stage case-control association study was used.In the first study,a small population comprising 401 Partridge chickens(201 cases and 200 controls)was used,and a total of 118 SNPs genotyped.In the second study,a bigger population comprising 1 075 Partridge chickens(527 cases and 548 controls) was used,and SNPs with significant effect determined in the first study were further analysed.In the first study,8 SNPs were significantly associated with S.Pullorum disease susceptibility/resistance,however,after further analysis,only the SNPs rs15001532 and C.513AT were found to be significantly associated with S.Pullorum disease susceptibility/resistance.The relative risk test demonstrated that the AA genotype of rs15001532 resulted in a higher risk of S.Pullorum infection,whereas birds with the TT genotype of C.513AT were more susceptible to S.Pullorum infection.This research was based on the researches on human complex diseases.With help of these train of thoughts,some common animal diseases can be studied effectively and the process of candidate gene research for animal disease can be improved.     

大肠杆菌“菌影”的制备

通过PCR扩增噬菌体PhiX174的裂解基因E,将该基因连接到含有PR/cI启动阻遏系统的pBV220载体中,从而使裂解基因E和启动阻遏系统λPL/PR-cI857串联成为温度敏感的裂解盒,构建溶菌质粒pBV-E。采用CaCl2法将其转入大肠杆菌BL21(DE3),含有裂解质粒的大肠杆菌在28℃条件下生长到对数生长期,然后升温到42℃诱导其溶解,制成了大肠杆菌菌影。电镜观察发现菌影形态完整,内容物全部被释放到胞外。     

Cellular immune responses of BALB/c mice induced by intramuscular injection of PRRSV-ORF5 DNA vaccine with different doses

BALB/c mice were immunized with 50 μg, 100 μg, 200 μg of pcDNA-PRRSV-ORF5 DNA vaccine respectively by intramuscular injection, with PBS and pcDNA3.1(+) as controls. Fluorescence activated cell Sorter (FACS) was used to detect the number of CD₄ ⁺ and CD₈ ⁺ T-lymphocytes. T-lymphocyte proliferation test was used to detect proliferation of the T-lymphocyte cells in peripheral blood lymphocytes of mice vaccinated with pcDNA-PRRSV-ORF5 DNA vaccine. The results showed that the difference in ConA response to T-lymphocytes in blood was highly significant between all experimental groups and the control group (P < 0.01). The number of CD₄ ⁺ T-lymphocytes in experimental groups was significantly higher than that of the control group 7 d after vaccination. The number of CD₈ ⁺ T-lymphocytes in the experimental groups was higher than that of the control group 28 d after vaccination. Mice immunized with a higher dose (200 μg) of DNA vaccine demonstrated higher cellular immune response than those immunized with a lower dose (100 μg, 50 μg) of DNA vaccine. The results demonstrated that pcDNA-PRRSV-ORF5 DNA vaccine could induce a good cellular immune response which may be dose-dependent. __________ Translated from China J Vet Sci Mar, 2006, 26(2): 111–114 [译自: 中国兽医学报] The first three authors contribute equally to this work.     

噬菌体裂解基因E原核表达载体构建及大肠杆菌菌影的制备

根据Genbank已知序列,设计1对引物,采用PCR方法扩增PhiX174噬菌体裂解基因E,经克隆、鉴定及序列测定分析,将该基因亚克隆到原核表达载体pGEX-6P-1,构建了能够在大肠杆菌中表达裂解基因E的载体pGEX-E,并采用CaCl2法将其转入大肠杆菌BL21(DE3),经诱导后成功获得了大肠杆菌菌影,为进一步研究菌影这一新型的疫苗制备方法和新的疫苗佐剂奠定了基础。     

鸡IL-18真核表达质粒与新城疫病毒F基因疫苗联合免疫探讨

为探讨鸡IL-18基因真核表达质粒pcDNA-chIL-18在新城疫病毒(NDV)F基因疫苗免疫中的作用,克隆了新城疫病毒标准株F48E9的F基因,构建F基因重组真核表达质粒pcDNA-F;将11日龄试验鸡分为4组,每组10只,14日龄一免,28日龄二免,分别肌注pcDNA-F、pcDNA-F+pcDNA-ckIL-18、新城疫传统疫苗(14日龄NDV弱毒疫苗首免,28日龄NDV油苗二免)、生理盐水,一免疫后第7、14、21、28 d均采血进行ND抗体(ELISA)检测和T淋巴细胞转化(MTT)试验;第29 d均肌注50LD50NDV强毒F48E9株.结果显示:免疫保护效率,生理盐水组0/10,pcDNA-F免疫组3/10,pcDNA-F+pcDNA-chIL-18免疫组5/10,新城疫传统疫苗免疫组10/10;ND抗体水平,pcDNA-F免疫组与pcDNA.F+pcDNA-chIL-18免疫组无差异(P>0.05),均显著低于传统疫苗免疫组(P<0.05);T细胞转化水平,pcDNA.F+pcDNA-cML-18免疫组明显高于pcDNA-F免疫组(P<0.05),pcDNA-F免疫组与传统疫苗免疫组无差异(P>0.05).试验结果综合显示pcDNA-F免疫试验鸡可产生一定程度的免疫保护;pcDNA-chIL-18对pcD-NA-F具有一定的免疫增强作用.     

EDSV六邻体蛋白基因核酸疫苗对减蛋综合征灭活油乳苗的免疫协同作用

利用所构建的减蛋综合征病毒六邻体蛋白基因真核表达质粒pcDNA3-hexon和减蛋综合征灭活油乳苗,观察了EDSV六邻体蛋白基因核酸疫苗对减蛋综合征灭活苗的免疫协同作用。结果显示,7日龄时应用核酸疫苗和减蛋综合征灭活油乳苗联合免疫的鸡群,在14~56日龄整个观察期内,胸腺T淋巴细胞对ConA、法氏囊B淋巴细胞对PMA的反应,均明显强于单纯油乳苗免疫组(其中,除在接种后第7天两组鸡的B淋巴细胞增殖OD570值差异不显著外,其余各检测时间两组间均表现为显著或极显著差异);特别是T淋巴细胞增殖情况在接种第14天后均表现为差异极显著。而且,HI抗体效价水平也持续性高于单纯灭活油乳苗免疫鸡群,并在免疫接种后35~49 d时差异显著。说明EDSV六邻体蛋白基因核酸疫苗与灭活油乳苗联合免疫,可明显提高机体的细胞免疫和体液免疫水平,产生很好的免疫协同作用。     

鸡白痢发生的微生态学机理的研究

本文运用微生态学原理和方法,通过对不同生理状态的7日龄雏鸡肠道中大肠杆菌、葡萄球菌、肠球菌、乳酸杆菌、拟杆菌、双歧杆菌、鸡白痢沙门氏杆菌进行了定量检测,从而阐明了雏鸡白痢病原菌与肠道常在优势菌的量变关系及其鸡白痢发生的微生态学机制。另外,还对鸡白痢的生态疗法进行了讨论。     

鸡传染性喉气管炎病毒gD基因在重组痘病毒中表达及其免疫效力

将鸡传染性喉气管炎病毒(ILTV) 河南长葛株gD基因重组到鸡痘病毒基因组中,获得了能高效表达糖蛋白D的重组鸡痘病毒(rFPV-gD) .将rFPV-gD经皮下刺种途径接种30日龄的固始鸡,用ILTV商品弱毒疫苗经滴鼻、点眼途径免疫试验鸡,同时设非免疫对照鸡群.分别于免疫后7、14、21、28、35和42 d采血分离血清,用微量血清中和试验测定ILTV抗体效价,并于免疫后42 d用ILTV强毒攻击所有试验鸡只.血清中和抗体检测结果表明:疫苗对照组与重组病毒组差异不显著(P>0.05) ,而重绀病毒组和疫苗对照组与空白对照组相比筹异均显著(P<0.05) .免疫42 d攻毒后鸡的发病情况表明,重组病毒组和疫苗对照组对ILTV强毒攻击的保护率均为96.67%,而空白对照组为6.67%,说明该重组病毒作为弱毒疫苗能够抵抗ILTV强毒的攻击,对免疫鸡群有较好的保护作用.     

中药“增免散”对鸡免疫功能及ND疫苗免疫应答的影响

本试验对中药免疫增强剂“增免散”对雏鸡新城疫(ND)疫苗免疫应答和免疫功能的影响进行了研究。1日龄艾维茵肉鸡健雏200只随机分4组,于7 d、21 d用La Sota活苗免疫,从第7天开始“增免散”按饲料量的0.5%、1.0%、1.5%添加,分别为Ⅰ、Ⅱ、Ⅲ组,Ⅳ组(对照组)不添加任何药物。各鸡群于7、14、28和49 d测定血液中新城疫血凝抑制(HI)抗体水平,并检测“增免散”对免疫功能的影响。结果表明,中药“增免散”按1%、1.5%添加能显著提高La Sota活苗接种雏鸡的特异性HI抗体水平,延长其持续时间,能促进雏鸡免疫器官的生长发育,能显著增高雏鸡外周血T淋巴细胞ANAE(Acida naphthyl acetate esterase)阳性率和B淋巴细胞EAC(Erythrocyte-antibody-Complement)花环率,并对雏鸡红细胞免疫粘附功能具有显著促进作用。     

鸡白细胞介素-18真核表达载体构建与ILTV弱毒疫苗联合免疫

为了探索鸡IL-18在传染性喉气管炎病毒(ILTV)弱毒疫苗中的免疫佐剂作用,将鸡IL-18(ChIL-18)克隆至真核表达载体pcDNA3.1(+)中,构建重组真核表达质粒pcDNA3.1/ChIL-18(简称pIL-18)。用pIL-18、ILTV弱毒疫苗及其两者联合分别免疫21日龄SPF雏鸡,免疫后每周抽取外周血及外周抗凝血,应用ELISA和流式细胞仪分别检测免疫鸡的体液免疫及细胞免疫水平。免疫后第4周用100 ELD50ILTV强毒进行攻击。结果pIL-18与ILTV弱毒疫苗联合免疫组的淋巴细胞亚群CD3+、CD3+CD4+、CD3+CD8+明显高于ILTV弱毒疫苗免疫组(P<0.01),ILTV强毒攻毒后发病鸡数量少,并且发病症状较轻。     

不同基因型NDV灭活油乳苗与La Sota株灭活油乳苗的免疫效果比较

用地方分离的基因Ⅵ型和Ⅶ型代表株 (LD - 3- 0 0株和LD - 7- 0 2株 )配以传统的LaSota株所制备的灭活油乳苗与LaSota株单价灭活油乳苗进行的免疫效果比较显示 :前者在接种后各个时期所产生的HI抗体效价与LaSota株灭活苗免疫鸡所产生的HI抗体效价无明显差异 ,至接种后第 4 2天两者均具有 7.3log2 的HI效价。在对具有不同HI抗体水平的试验鸡进行的攻毒试验中 ,对F4 8E9标准强毒株的攻击 ,无论是LaSota株疫苗免疫鸡 ,还是不同基因型灭活苗免疫鸡 ,在HI抗体效价为 6log2 以上时均能 10 0 %地被保护 ;但对不同基因型野毒株的攻击 ,尤其是基因Ⅶ型野毒株的攻击 ,2种疫苗则呈现出明显的差异。在LaSota株疫苗免疫鸡HI效价为 6log2 时 ,对不同基因Ⅶ型野毒株的攻击 ,保护率只有 0～ 2 0 % ;而具有 6log2 效价的不同基因型疫苗免疫鸡则对不同基因Ⅶ型野毒株的攻击具有 6 0 %～ 70 %的保护率 ;当HI效价达 8log2 左右时 ,LaSota株疫苗免疫鸡对基因Ⅶ型野毒株的攻击也只有 80 %的保护率 ,而不同基因型疫苗免疫鸡已能 10 0 %地被保护 ;LaSota株疫苗免疫鸡只有在HI效价在 9log2 以上时 ,方可 10 0 %地抵抗基因Ⅶ型野毒株的攻击。     

Evaluating the efficacy of an attenuated Streptococcus equi ssp. zooepidemicus vaccine produced by multi-gene deletion in pathogenicity island SeseCisland_4

Streptococcus equi ssp. zooepidemicus(SEZ) is a pathogen associated with a wild range of animal species. Frequent outbreaks have occurred in recent years in pigs, horses, goats and dogs which is liable to infect humans. There is a lack of efficient vaccines against this disease and the occurrence of antibiotic resistance may render drug therapies ineffective. In this study, gene deletion mutant(ΔSEZ) in pathogenicity islands SeseCisland_4 was constructed. The mutant ΔSEZ had a 52-fold decrease in 50% lethal dose(LD_(50)) and had less capacity to adhere epithelial cells. Importantly, immunization of mice with attenuated vaccine ΔSEZ at the dose of 10~2 colony-forming units(CFU) mL~(–1) elicited a significant humoral antibody response, with an antibody titer of 1:12 800. Therefore, 10~2 CFU mL~(–1) might be used as the appropriate immune dose for the attenuated vaccine ΔSEZ, which provided mice with efficient protection against virulent SEZ. In addition, the hyperimmune sera against 10~2 CFU m L–1 attenuated vaccine ΔSEZ could confer significant protection against virulent SEZ infection in the passive immunization experiment and exhibited efficient bactericidal activity in the whole blood assay. Meanwhile, no viable bacteria was detected in blood when mice were immunized with ΔSEZ at the dose of 10~2 CFU mL~(–1) via hypodermic injection. Thereafter, the mutant ΔSEZ at the dose of 10~2 CFU mL~(–1) could confer significant protection in mice and had less negative effects on host, which could be an effective attenuated vaccine candidate for the prevention of SEZ.     

新城疫和传染性法氏囊病二联疫苗的免疫效力研究

以LaSota系新城疫(ND)病毒制成ND弱毒疫苗,以含传染性法氏囊病(IBD)病毒VP2基因重组质粒pET28a/VP2的大肠杆菌BL21制成IBD重组活载体疫苗,将ND弱毒疫苗和IBD重组活载体疫苗组建ND-IBD二联疫苗.将试验鸡分成8个组,分别肌肉注射接种ND弱毒疫苗、IBD重组活栽体疫苗和ND-IBD二联疫苗.最后一次接种后第10天,对所有试验鸡进行采血,分离血清,以病毒血凝抑制试验(HI)及琼脂免疫扩散试验检测NDV抗体和IBDV抗体.对NDV HI抗体阳性率进行x2检验及对NDV HI效价进行方差分析,结果对照组与其他4组之间差异均显著(P＜0.05),而试验组(B、C、D、E)4组之间差异均不显著(P＞0.05).对IBDV抗体阳性率进行x2检验及对IBDV沉淀抗体滴度进行方差分析,结果在A、B、F 3组之间差异不显著(P＞0.05),在C、D、G、H 4组之间差异不显著(P＞0.05);而A、B、F 3组与C、D、G、H 4组之间差异显著(P＜0.05).试验表明,在诱导雏鸡体液免疫应答方面,ND-IBD二联疫苗与ND弱毒疫苗和IBD重组活载体疫苗具有同样的效力.     

不同日龄鸡呼吸道黏膜中IgA分泌细胞的分布规律

目的 了解鸡呼吸道黏膜不同时期的免疫状态。方法 选择海兰白鸡鸡胚(18、20 日龄)及不同日龄(1、4、7、14、21、35和56 日龄)雏鸡的鼻、喉、气管和肺脏样品,利用免疫组织化学方法研究IgA⁺分泌细胞的出现、定位分布及数量变化过程。结果 呼吸道各个器官黏膜中胚胎时期均没有IgA⁺细胞出现,鼻和肺脏中1日龄时出现,喉黏膜中4日龄时出现,而气管黏膜中7日龄时才出现。随着日龄增长,各器官呼吸道黏膜中IgA⁺细胞数量均逐渐增加。鼻黏膜以及肺脏初级支气管和次级支气管交叉处4日龄时较早地形成淋巴聚集物,喉黏膜中7日龄时形成,气管黏膜中35日龄时形成。在这些淋巴聚集物中IgA⁺细胞均主要分布于淋巴聚集物的外周。35日龄时,在鼻、喉和气管黏膜固有层以及上皮内均存在较多的IgA⁺细胞,从而能够更有效和直接地分泌SIgA,执行黏膜免疫功能。56日龄时,4种器官的呼吸道黏膜中IgA⁺细胞的数量达到峰值,并在黏膜底部形成生发中心,具有了黏膜相关性淋巴组织的典型特征,从而更有效地执行黏膜免疫功能。结论 鸡呼吸道黏膜中IgA⁺细胞的分布和数量均呈现日龄相关性变化,并且在35日龄时,鼻、喉、气管和肺脏中IgA⁺细胞数量和分布部位均达到一定规模,能够为呼吸道黏膜提供免疫保护。     

Baculovirus-expressed FAdV-4 penton base protein protects chicken against hepatitis-hydropericardium syndrome

Hepatitis-hydropericardium syndrome (HHS) is an infectious disease caused by fowl adenovirus serotype 4 (FAdV-4). Several structural and non-structural proteins of FAdV-4 have been expressed in Escherichia coli and baculovirus expression system to develop candidate subunit vaccines. However, the protective efficiency of baculovirus-expressed penton base protein has not been assessed. In this study, two recombinant capsid proteins, penton base and fiber-2, were constructed. And then, penton base and fiber-2 were administrated alone or together to specific pathogen-free (SPF) chickens at 14 days of life and boosted at 28 days of life. At 42 days of life, the immunized groups and the control group were challenged with FAdV-4 virulent strain. Results show that inoculating penton base or penton base+fiber-2 provided 100% protection to the chickens. All groups vaccinated with the recombinant protein produced detectable antibodies and showed no apparent lesions. Thus, baculovirus-expressed penton base protein is a promising candidate subunit vaccine.     

表达载体pCAGGS显著增强禽流感DNA疫苗的免疫保护效果

【目的】将A/Goose/GuangDong/1/96(H5N1)[GD/1/96(H5N1)]的HA基因插入鸡β-actin启动子高效真核表达载体pCAGGS，构建了DNA疫苗质粒pCAGGHA5，以提高H5亚型禽流感DNA疫苗的表达水平和免疫保护效果。【方法】将pCAGGHA5和表达GD/1/96(H5N1)HA基因的质粒pCIHA5通过间接免疫荧光法和Western-blot分析检测转染293T细胞后瞬时表达的HA抗原蛋白，随之将pCAGGHA5及pCIHA5分别以100 ?g和10 ?g剂量一次免疫3周龄SPF鸡，4周后用100 LD50的HPAIV GD/1/96(H5N1)鼻腔途径进行攻击。【结果】间接免疫荧光法和Western-blot分析表明2种表达质粒均可正确表达H5亚型HA抗原蛋白，载体pCAGGS表达水平显著高于载体pCI；免疫SPF鸡后，100?g pCAGGHA5可形成5/5的免疫保护，100?g pCIHA5可形成2/4的免疫保护，10?g pCAGGHA5可形成对免疫鸡5/5的免疫保护，而10?g pCIHA5 则基本不能形成免疫保护，pCAGGHA5诱导的HI抗体水平远远高于pCIHA5。【结论】鸡β-actin启动子表达载体pCAGGS可显著提高HA基因体外表达水平和H5亚型禽流感DNA疫苗诱导的保护性抗体免疫反应水平，增强免疫保护效果。     

鸡蛋黄抗体(IgY)的分离与研究

以鸡白痢沙门氏菌灭活苗,鸡新城疫(NDV)油剂灭活苗,传染性法氏囊炎(IBDV)油剂灭活苗,分别免疫接种健康母鸡.用异丙醇——丙酮法,分离提取高免鸡鸡蛋的蛋黄抗体(IgY)·经凝集试验,血球凝集抑制试验(HI)和琼脂扩散试验(AGP)检测蛋黄抗体(IgY)的抗体滴度,结果分别均高于血清的抗体滴度.另外,还以聚丙烯酰胺凝胶电泳(SDS-PAGE)监测比较了蛋黄抗体(IgY),血清和蛋黄液,结果表明,IgY的抗体球蛋白纯度均高于血清和蛋黄液.