藏鸡和隐性白羽鸡感染柔嫩艾美耳球虫后免疫相关基因的表达变化分析

试验旨在从免疫遗传学的角度初步探讨藏鸡（TC）和隐性白羽鸡（RWC）对柔嫩艾美耳球虫（Eimeria tenella）易感性差异的分子机制,分别用1×10⁵个柔嫩艾美耳球虫孢子化卵囊感染对球虫具有抗性的藏鸡和易感的隐性白羽鸡。应用实时荧光定量PCR检测藏鸡和隐性白羽鸡感染前0 d和感染后第2、4、6和8天脾脏、盲肠、胸腺、法氏囊中γ-干扰素（IFN-γ）、白细胞介素-2（IL-2）、IL-16、Toll样受体（TLR3）和TLR15免疫相关基因的转录水平变化。结果显示,藏鸡脾脏IFN-γ、IL-2、IL-16及TLR3、TLR15免疫相关基因转录水平于感染后第4和8天明显上调,隐性白羽鸡则无明显变化。藏鸡盲肠IFN-γ转录水平在感染后第2天显著上调（P<0.05）,TLR3在感染后第4天起显著上调（P<0.05）,其余免疫相关基因变化幅度不大;隐性白羽鸡盲肠IFN-γ转录水平在感染后第8天显著上调（P<0.05）,IL-2在感染后第2天起显著上调（P<0.05）,IL-16在感染后第6天起显著上调（P<0.05）,TLR3在感染后第2和8天显著上调（P<0.05）,TLR15变化幅度不大。各免疫相关基因在2个品种鸡胸腺和法氏囊中均出现上调或下调,但除藏鸡法氏囊TLR3和TLR15转录水平变化幅度相对较大外,其余免疫相关基因与感染前相比变化幅度不大。以上结果显示,球虫感染主要导致藏鸡和隐性白羽鸡脾脏和盲肠中的各免疫相关基因出现显著变化,表明宿主的遗传背景在一定程度上可影响球虫感染的免疫应答。     

人工感染柔嫩艾美耳球虫对雏鸡盲肠正常菌群的影响

在对雏鸡人工感染柔嫩艾美耳球虫后6、8、13、20天,分别对感染雏与未感染雏盲肠内容物中梭杆菌、真杆菌、消化球菌、乳酸杆菌、类杆菌、肠球菌、葡萄球菌和肠杆菌等8种主要正常菌群进行定性、定量分析。结果发现在球虫感染第20天,感染组有益菌如乳酸杆菌的数量低于对照组,差异极显著(P<0.01),有害菌如梭杆菌、葡萄球菌数量感染组高于对照组,其中梭杆菌差异显著(P<0.05),葡萄球菌差异极显著(P<0.01)。     

中药"球康"对E.tenella感染(鸡)IgA抗体免疫应答的影响

本试验通过检测柔嫩艾美耳球虫(E.tenella)感染鸡的血清和盲肠液中免疫球蛋白A(IgA)的含量变化,来分析中药“球康”的抗球虫作用效果。结果表明,中药“球康”能增强柔嫩艾美耳球虫感染鸡的IgA免疫应答反应,但是效果不明显。     

柔嫩艾美尔球虫(E.tenella)感染鸡IgG生成细胞及血清IgG变化

为了探明鸡球虫保护性免疫机制,通过人工复制的鸡球虫病鸡,利用免疫组化技术和斑点酶联免疫吸附试验(Dot-ELISA),分别检测了柔嫩艾美耳球虫(E.tenella)初次感染雏鸡后,盲肠局部和免疫器官中IgG生成细胞数的动态变化,循环血液中特异性IgG水平的动态变化;雏鸡母源抗体的动态变化和不同抗体水平雏鸡的抗球虫能力.结果表明:(1)攻毒后雏鸡盲肠粘膜、脾脏、法氏囊、盲肠扁桃体中的IgG生成细胞早于第2～3d(d)开始增殖,在第9～12d达到峰值,随后即开始下降,盲肠扁桃体中的IgG生成细胞数在第22d仍高于对照组.(2)雏鸡感染E.tenella后第6d即可在循环血液中检测到特异性IgG,于第18d达到峰值,第30d降至感染后第7d时的水平.(3)特异性母源抗体IgG水平高的雏鸡,抗球虫水平高.雏鸡母源抗体IgG水平随日龄增长逐渐下降,同时雏鸡的抗球虫水平也随之降低,母源抗体IgG水平与抗球虫能力有明显的正相关性.     

感染E.tenella对肉仔鸡生长、营养物质表观存留率及血液生理指标的影响

为了研究玉米 -豆粕型基础饲养下感染球虫对肉仔鸡生长、营养物质表观存留率、血液生理指标的影响 ,用 48只 1周龄 ( AA)肉仔鸡进行试验 ,按体重随机分为 2组 ,代谢期 1周。试验组鸡 1 4日龄时分别感染柔嫩艾美耳球虫卵囊 9万个 /只。结果表明 :试验组除饲料转化比 ,在代谢期后 4天间有显著差异外 ,其余多数生长性能指标在两组间无显著差异 ( P>0 .1 )。但感染球虫降低了试验组鸡的营养物质的表观存留率     

rChlFN-γ对E.tenella卵囊免疫雏鸡肠道分泌型IgA+细胞数量的影响

本研究初步探讨了重组鸡γ干扰素(rChIFN-γ)对柔嫩艾美耳球虫(E.tenella)卵囊免疫接种鸡肠道粘膜slgA+细胞数量的影响.分别给7和14日龄雏鸡予以E.tenella孢子化卵囊免疫接种并同时肌注5 000 urChlFN-γ后,于21日龄用同源E.tcnella攻虫.免疫组化检测结果表明rChIFN-γ加强免疫组在21日龄时十二指肠、空肠、盲肠和回肠的绒毛sIgA+细胞数量均高于免疫对照组,在28日龄时,均呈现显著性差异(α=0.05).结果表明rChIFN-γ可提高球虫卵囊免疫雏鸡肠道内的sIgA+细胞数量,增强粘膜免疫水平,具有明显的抗球虫免疫增强效果.     

雏鸡感染柔嫩艾美耳球虫后脂质过氧化物的变化

为了探讨柔嫩艾美耳球虫 (E.tenella)对感染雏鸡的损伤机制 ,本试验给 10日龄海兰白公雏鸡口服柔嫩艾美耳球虫孢子化卵囊 8× 10 4个 /只 ,在感染后 2、4、6、7、9、12、16和 2 1d分别各取 5只雏鸡剖检 ,取盲肠并采血 ,观察盲肠病理组织学变化 ,测定盲肠组织及血清中的丙二醛 (MDA )、超氧化物歧化酶 (SOD)、谷胱甘肽过氧化物酶 (GSH- Px)的水平。结果表明 ,雏鸡在感染 E.tenella后 2～ 4d,盲肠开始出现损伤 ,同时 SOD和 GSH- Px活性也开始下降 ;感染后 4～ 7d,盲肠粘膜上皮和肠腺上皮细胞大量变性、坏死崩解 ,发生严重损伤时 ,盲肠及血清中 MDA含量显著增加 ,并持续到 9d,同时 SOD和 GSH- Px活性也显著下降 ;感染 9d后 ,损伤的盲肠组织及 MDA、SOD和 GSH- Px水平逐渐恢复 ,至 2 1d基本达到正常     

柔嫩艾美耳球虫Etron2基因在不同发育阶段转录水平差异的分析

为研究柔嫩艾美耳球虫不同发育时期Etron2基因的转录水平差异,选取E.tenella生活史中的未孢子化卵囊、孢子化卵囊、子孢子、第二代裂殖子,分别提取总RNA,反转录为cDNA;选择Etactin作为参照基因,Etron2为目的基因,设计实时荧光定量PCR的引物,分析在E.tenella生活史中不同发育阶段Etron2转录水平的差异。结果显示Etron2在未孢子化卵囊中转录水平最高,然后分别是裂殖子、孢子化卵囊和子孢子。在柔嫩艾美耳球虫发育过程中,Etron2基因可能在未孢子化卵囊阶段被转录储备,随着宿主细胞的刺激,得以大量表达,在入侵过程中发挥作用。     

柔嫩艾美尔球虫（E．tenella）感染鸡IsG生成细胞及血清IgG变化

为了探明鸡球虫保护性免疫机制，通过人工复制的鸡球虫病鸡，利用免疫组化技术和斑点酶联免疫吸附试验（Dot-ELISA），分别检测了柔嫩艾美耳球虫（E．tenella）初次感染雏鸡后，盲肠局部和免疫器官中IgG生成细胞数的动态变化，循环血液中特异性IgG水平的动态变化；雏鸡母源抗体的动态变化和不同抗体水平雏鸡的抗球虫能力。结果表明：（1）攻毒后雏鸡盲肠粘膜、脾脏、法氏囊、盲肠扁桃体中的IgG生成细胞早于第2～3d（d）开始增殖，在第9～12d达到峰值，随后即开始下降，盲肠扁桃体中的IgG生成细胞数在第22d仍高于对照组。（2）雏鸡感染E．tenella后第6d即可在循环血液中检测到特异性IgG，于第18d达到峰值，第30d降至感染后第7d时的水平。（3）特异性母源抗体IgG水平高的雏鸡，抗球虫水平高。雏鸡母源抗体IgG水平随日龄增长逐渐下降，同时雏鸡的抗球虫水平也随之降低，母源抗体IgG水平与抗球虫能力有明显的正相关性。     

柔嫩艾美耳球虫抗药性相关基因M20的克隆与表达

利用前期构建的柔嫩艾美耳球虫（Eimeria tenella）地克珠利抗药株和马杜拉霉素抗药株与各自母株之间的抑制性消减文库而获得的ESTs序列,选择其中一个差异表达的EST序列（克隆号为M20）,采用RACE技术,以柔嫩艾美耳球虫敏感株孢子化卵囊cDNA为模板,扩增获得了该基因的全长cDNA序列,命名为M20,该基因全长847 bp,包括一个525 bp的开放阅读框,编码174个氨基酸,预测理论分子量约为19 ku。实时荧光定量PCR结果显示,该基因在敏感株中表达量高于地克珠利抗药株和马杜拉霉素抗药株;在不同发育阶段中,未孢子化卵囊表达量高于孢子化卵囊、子孢子以及裂殖子阶段虫体。将该基因克隆于原核表达质粒pET28b中,构建重组质粒pET28b-M20,转化大肠杆菌BL21（DE3）后,经IPTG诱导表达获得了重组蛋白,分子量约约为23 ku,该蛋白以包涵体形式存在,在IPTG诱导8 h后表达稳定,Western blot检测表明,其具有较好的免疫原性。     

鸡盲肠扁桃体组织结构早期发育的动态观察

本研究利用HE染色方法结合图像分析软件和数据统计软件,观察雏鸡不同生长发育阶段盲肠扁桃体的组织学发育过程和结构特征。结果显示:随着日龄的增长,盲肠扁桃体特征结构不断发育成熟,并在21日龄时基本达到成熟水平。21日龄时,盲肠扁桃体粘膜层中下部形成生发中心,其数目在35日龄时达到稳定。证实,鸡出壳后初期,盲肠扁桃体免疫功能迅速增强,并在35日龄时达到成熟水平。     

Development of Peyer's patch and cecal tonsil in gut-associated lymphoid tissues in the chicken embryo

It is well known that chicken B cells develop in the bursa of Fabricius (BF), which is categorized as gut-associated lymphoid tissue (GALT). Chicken GALT also includes Peyer's patch (PP) and cecal tonsil (CT). The relationship between these tissues in GALT during B cell development is currently unknown. In this study, we conducted comparative examination of PP, CT and BF development during embryogenesis using immunohistochemical staining. On day 13 of embryogenesis (E13), accumulation of MHC class II(+) cells was observed in the intestine. Thereafter, Bu-1(+) cells and IgM(+) cells appeared, and their number continuously increased at the same sites where MHC class II(+) cells were present. Similar results were obtained in the CT. The locations of embryonic PP were limited to two sites; near the Meckel's diverticulum and the ileocecal junction. Anlage of bursal follicles first appeared at E13 and developed thereafter. Immigration of Bu-1(+) cells to bursal follicles began at E13, and the number of Bu-1(+) cell subsequently increased. When the follicle of BF was eliminated from the embryo by treatment with testosterone, development of PP and CT were observed. We concluded therefore that the development of PP and CT start during late embryogenesis at the same time as the follicle of BF, and that appearance of surface IgM(+) cells in PP and CT is independent form the development of the follicle of BF.     

Cytokine gene expression in chicken cecal tonsils following treatment with probiotics and Salmonella infection

Probiotics are currently employed for control of pathogens and enhancement of immune response in chickens. In this study, we investigated the underlying immunological mechanisms of the action of probiotics against colonization of the chicken intestine by Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella serovar Typhimurium). Birds received probiotics by oral gavage on day 1 of age and, subsequently, received Salmonella serovar Typhimurium on day 2 of age. Cecal tonsils were removed on days 1, 3 and 5 post-infection (p.i.), RNA was extracted and subjected to real-time quantitative RT-PCR for measurement of interleukin (IL)-6, IL-10, IL-12 and interferon (IFN)-gamma gene expression. There was no significant difference in IL-6 and IL-10 gene expression in cecal tonsils of chickens belonging to various treatment groups. Salmonella serovar Typhimurium infection resulted in a significant increase in IL-12 expression in cecal tonsils on days 1 and 5p.i. However, when chickens were treated with probiotics prior to experimental infection with Salmonella, the level of IL-12 expression was similar to that observed in uninfected control chickens. Treatment of birds with probiotics resulted in a significant decrease in IFN-gamma gene expression in cecal tonsils of chickens infected with Salmonella compared to the Salmonella-infected birds not treated with probiotics. These findings reveal that repression of IL-12 and IFN-gamma expression is associated with probiotic-mediated reduction in intestinal colonization with Salmonella serovar Typhimurium.     

密码子优化的鸡白细胞介素-17在昆虫细胞中的表达研究

为了获得具有良好生物学活性且具有较高表达量的鸡白细胞介素-17(ChIL-17),本研究在前期研究工作基础上,将ChIL-17的编码基因按照真核细胞(昆虫细胞)偏爱的密码子进行优化改造,经全基因合成后插入到转座载体pFastBacTM Ⅰ中,构建重组转移质粒pfast-mod.ChIL-17并转化DH10Bac感受态细胞.通过位点特异性转座将mod.ChIL-17基因整合到穿梭质粒Bacmid中,获得重组穿梭质粒Bacmid-mod.ChIL-17.应用脂质体将重组穿梭质粒转染Sf9昆虫细胞,获得重组杆状病毒rBac-mod.ChIL-17.重组病毒传代扩增感染Sf9细胞,通过间接免疫荧光检测目的蛋白的表达.结果表明,经过优化的ChIL-17在杆状病毒系统中获得表达.     

Tissue expression and developmental regulation of chicken cathelicidin antimicrobial peptides

ABSTRACT: Cathelicidins are a major family of antimicrobial peptides present in vertebrate animals with potent microbicidal and immunomodulatory activities. Four cathelicidins, namely fowlicidins 1 to 3 and cathelicidin B1, have been identified in chickens. As a first step to understand their role in early innate host defense of chickens, we examined the tissue and developmental expression patterns of all four cathelicidins. Real-time PCR revealed an abundant expression of four cathelicidins throughout the gastrointestinal, respiratory, and urogenital tracts as well as in all primary and secondary immune organs of chickens. Fowlicidins 1 to 3 exhibited a similar tissue expression pattern with the highest expression in the bone marrow and lung, while cathelicidin B1 was synthesized most abundantly in the bursa of Fabricius. Additionally, a tissue-specific regulatory pattern was evident for all four cathelicidins during the first 28 days after hatching. The expression of fowlicidins 1 to 3 showed an age-dependent increase both in the cecal tonsil and lung, whereas all four cathelicidins were peaked in the bursa on day 4 after hatching, with a gradual decline by day 28. An abrupt augmentation in the expression of fowlicidins 1 to 3 was also observed in the cecum on day 28, while the highest expression of cathelicidin B1 was seen in both the lung and cecal tonsil on day 14. Collectively, the presence of cathelicidins in a broad range of tissues and their largely enhanced expression during development are suggestive of their potential important role in early host defense and disease resistance of chickens.     

Tissue expression and developmental regulation of chicken cathelicidin antimicrobial peptides

Cathelicidins are a major family of antimicrobial peptides present in vertebrate animals with potent microbicidal and immunomodulatory activities. Four cathelicidins, namely fowlicidins 1 to 3 and cathelicidin B1, have been identified in chickens. As a first step to understand their role in early innate host defense of chickens, we examined the tissue and developmental expression patterns of all four cathelicidins. Real-time PCR revealed an abundant expression of four cathelicidins throughout the gastrointestinal, respiratory, and urogenital tracts as well as in all primary and secondary immune organs of chickens. Fowlicidins 1 to 3 exhibited a similar tissue expression pattern with the highest expression in the bone marrow and lung, while cathelicidin B1 was synthesized most abundantly in the bursa of Fabricius. Additionally, a tissue-specific regulatory pattern was evident for all four cathelicidins during the first 28 days after hatching. The expression of fowlicidins 1 to 3 showed an age-dependent increase both in the cecal tonsil and lung, whereas all four cathelicidins were peaked in the bursa on day 4 after hatching, with a gradual decline by day 28. An abrupt augmentation in the expression of fowlicidins 1 to 3 was also observed in the cecum on day 28, while the highest expression of cathelicidin B1 was seen in both the lung and cecal tonsil on day 14. Collectively, the presence of cathelicidins in a broad range of tissues and their largely enhanced expression during development are suggestive of their potential important role in early host defense and disease resistance of chickens.     

鸡白介素17A的克隆表达及活性测定

从经人工感染柔嫩艾美尔球虫孢子化卵囊(1×104个/只鸡)的盲肠上皮间淋巴细胞(IELs)提取总RNA,用RT-PCR方法成功扩增了鸡白介素17A(IL-17A)基因,测序结果显示,开放阅读框为453bp,编码150个氨基酸,与GenBank上鸡IL-17A基因(AM773756)的核苷酸和氨基酸序列完全一致(100%),与报道的哺乳动物IL-17A的氨基酸相似性达37%～46%。构建的pGEX-6p1-chIL-17A原核表达载体经1mmol/L IPTG诱导后表达出43kDa左右的融合蛋白,与预期大小一致,且Western-blot鉴定表达产物为目的蛋白。功能试验表明,表达的重组鸡IL-17A能够刺激鸡胚成纤维细胞产生IL-6。这些结果表明,鸡IL-17A在结构和功能方面与哺乳动物的IL-17A都有一定的相似性,可能在鸡的免疫应答过程中起到重要的作用。     

鸡IL-2基因的克隆及GST-chIL2融合蛋白的表达

根据Sundick等发表的鸡IL-2基因(chIL2)序列设计合成特异性引物,用RT-PCR从ConA诱导的鸡脾淋巴细胞扩增出450 bp的目的片段,酶切鉴定及序列测定结果表明为鸡IL-2基因。该基因包括鸡IL-2基因的全部开放阅读框,编码142个氨基酸组成的蛋白质,与GenBank鸡IL-2基因相比,在编码氨基酸的49位有一个氨基酸缺失;而与Broiler、SC、Chenren和Xiaoshan鸡在编码氨基酸上完全一致,具有较近的亲缘关系;与Kestrel来航鸡、来航SPF鸡、Obese、Silky和Xianju鸡等有1-4个氨基酸的差异;与火鸡和鹌鹑的氨基酸同源性分别为69.9%和59.4%。将克隆到的基因插入到融合蛋白原核表达载体pGEX-6p-1中,得到重组表达质粒pGEX-IL2。将此重组质粒转化大肠杆菌DH5α,经IPTG诱导,表达出了大小约为40 ku的GST-chIL2融合蛋白,其中GST部分为26 ku,鸡IL-2为14 ku,与预期的鸡IL-2成熟蛋白大小一致。     

一起鸡传染性喉气管炎与大肠杆菌混合感染的诊治

鸡传染性喉气管炎为冬春季节多发病,随着外界温度的变化使鸡只产生应激反应导致抵抗力降低而发病。此病发病急,传播迅速,死亡率高,常与大肠杆菌病、支原体病等混合感染,严重危害了养鸡业的发展。现将一起诊治情况报告如下：     

维生素A和维生素E水平对北京鸭前期生产性能的影响

本试验采用5×2完全随机试验设计,5个维生素A(VA)添加水平(0、2500、5000、10000IU/kg和15000IU/kg),2个维生素E(VE)添加水平10mg/kg和100mg/kg,360只1日龄北京鸭随机分为10个处理组,研究不同水平VA、VE对北京鸭生产性能的影响。结果表明,VA的添加可显著提高北京鸭生长前期的平均日增重和平均日采食量(P0.01);对料肉比的影响差异不显著(P0.05),VE水平对北京鸭前期生产性能无显著影响(P0.05);建议北京鸭生长前期VA的适宜添加量为5000～10000IU/kg,VE为10mg/kg。