柔嫩艾美耳球虫卵囊免疫后鸡盲肠扁桃体GM-CSF基因表达的动态变化

为检测和评价鸡粒细胞巨噬细胞集落刺激因子（ChGM-CSF）在抗球虫免疫过程中的作用,以RT-PCR技术从鸡盲肠扁桃体中克隆获得了ChGM-CSF编码基因的开放阅读框（ORF）,其长为435 bp。将试验鸡随机分为对照组和试验组,试验组于12、22日龄时用E. tenella孢子化卵囊进行免疫,分别于设定时间每次取5只鸡采集盲肠扁桃体,以鸡β-actin为参照建立半定量RT-PCR方法检测ChGM-CSF的表达动态。结果表明,在第1次免疫后的第1天（13日龄,P＜0.05）和第2次免疫后的第7天（29日龄,P＜0.01）,试验组ChGM-CSF的表达量显著高于对照组;在第1次免疫后的第9天（21日龄）和第2次免疫后的第3天（25日龄）ChGM-CSF的表达量显著低于对照组（P＜0.01）。本研究提示ChGM-CSF参与了鸡体的抗球虫感染过程,为进一步探讨ChGM-CSF的生物学活性及作为鸡球虫病基因工程疫苗佐剂的研究奠定基础。     

柔嫩艾美球虫免疫与鸡盲肠扁桃体TNF-α基因表达动态的关系

根据GenBank上鸡β-actin、TNF-α的基因序列设计引物，应用逆转录-聚合酶链式反应（RT—PCR）技术克隆获得了pactin和TNF-α基因，采用pactin为内参的半定量方法检测TNF-αmRNA在鸡柔嫩艾美球虫免疫前后不同时间的表达情况，以探讨TNF-α基因的表达动态与柔嫩艾美球虫免疫的关系。结果显示，TNF-α基因在两次免疫期间的表达量整体上呈现双峰模式，首免后第9d达到一个高峰，二免后第7d达到另一个高峰。结果表明，TNF-α在抗球虫感染中有一定的作用。     

柔嫩艾美耳球虫免疫期间鸡盲肠扁桃体γ干扰素表达动态的检测

根据GenBank鸡β-actin、IFN-γ的基因序列设计引物,应用逆转录-聚合酶链反应（RT-PCR）技术克隆获得β-actin和IFN-γ基因,采用β-actin为内参的半定量方法检测IFN-γ mRNA在鸡柔嫩艾美耳球虫免疫前后不同时间的表达情况,以探讨IFN-γ的表达动态与柔嫩艾美耳球虫免疫的关系。结果显示,IFN-γ在两次免疫期间的表达量整体上呈现双峰模式,首免后第7天达到一个高峰,二免后第7天达到另一个高峰。     

柔嫩艾美耳球虫病鸡盲肠上皮细胞凋亡的分析

从细胞凋亡的角度探讨柔嫩艾美耳球虫(E.tenella)对鸡盲肠黏膜的损伤机理,为鸡球虫病的防治提供理论依据.用E.tenella孢子化卵囊感染雏鸡,于感染后不同时间段取盲肠组织,运用常规病理组织学、超微病理学和原位末端标记技术对感染E.tenella病鸡肓肠黏膜上皮细胞和肠腺上皮细胞进行观察和分析.结果表明:鸡感染E.tenella的第2天,盲肠黏膜上皮细胞凋亡数即开始增加,感染后第4～6天是病变最严重且具有特征性的阶段,主要表现为中、后段盲肠黏膜深层严重出血,大最黏膜上皮脱落,严重者可见整个黏膜几乎完全脱落,肠腺破坏,其表面被覆盖大量脱落的变性、坏死的上皮细胞和红细胞,残存的黏膜上皮细胞和肠腺上皮细胞凋亡速度加快,大多数上皮细胞处于凋亡状态,凋亡指数极显著(P≤0.01)高于空白对照组,凋亡细胞体积缩小,细胞线粒体肿胀,严重时破裂成大空泡,染色质致密边集或断裂成核碎片,形成膜包裹性凋亡小体.结果提示E.tenella感染鸡后盲肠上皮细胞凋亡加重,以裂殖生殖阶段最为明项,且与损伤程度和病程密切相关.     

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。     

NO对柔嫩艾美耳球虫感染鸡盲肠粘膜大细胞及组胺的影响

将试验鸡分为5组，每组18只，除空白对照组外，其他组鸡均用柔艾美耳球虫（E.tenella)进行免疫和攻毒接种，其中3组分别给予氨基胍（AG），N^W-硝基－L－精氨酸甲酯（L－NAME）和L-精氨酸（NOS底物，L－Arg）处理，结果表明，血清中NO^-2浓度以AG处理组最低，L－NAME处理组和L－Arg处理组比空白对照组明显升高，但都显著低于免疫攻毒组（P＜0．05），AG处理组盲肠肥大细胞（MC）数与空白对照组无明显差异，而极显著地高于其他组（P＜0．01），免疫攻毒组盲肠MC数明显高于L－NAME处理组和L－Arg处理组，而L－NAME处理组与L－Arg处理组之间则没有明显差异，AG处理组鸡盲肠组胺（HT）含量极显著高于其他组（P＜0．01）；LAME处理和L－Arg处理组的HT含量差异不显著，但都高于空白对照组而你于免疫攻毒组。体外试验明，磷酸组织胺能够明显抑制伴刀豆球蛋白A对鸡外周血淋巴细胞的诱导增殖作用（P＜0．01），结果提示，E.tenella感染鸡盲肠MC数与HT含量之间未见明显的相关性，活化的MC或其分泌物HT和NO产生有一定抑制作用，另一方面，NO也可能抑制MC的增殖。     

柔嫩艾美耳球虫细胞培养液对鸡胚盲肠上皮细胞活性的影响

为了探讨鸡柔嫩艾美耳球虫(E.tenella)分泌物及代谢产物对鸡胚盲肠上皮细胞活性的影响,用E.tenella裂殖生殖期细胞培养液培养鸡胚盲肠上皮细胞24 h,通过MTT法检测细胞活性,HE染色观察细胞形态变化。结果:用含25%的48 h及72 h攻虫培养液的混合培养基可使鸡盲肠上皮细胞的活性显著下降(P0.01)。表明E.tenella裂殖生殖期的分泌物及代谢产物可降低未感染鸡胚盲肠细胞的活性。     

柔嫩艾美耳球虫感染鸡盲肠和脾脏一氧化氮合酶的表达

采用NADPH—d(nicotinamide adenine dinucleotide phosphate—diaphroase)组织化学法观察了雏鸡感染柔嫩艾美耳珠虫(E．tenella)后一氧化氮合酶(N0S)在盲肠和脾脏中的分布与表达情况。试验结果表明，所有正常鸡盲肠粘膜下层和肌层均有较深的着色，根据以往的资料和N0S的表达特性初步判断为神经元型N0S(nNOS)；试验鸡在感染后3～5d，盲肠粘膜上皮和肠腺上皮也有较深的着色，并从感染后7d开始着色减弱；而对照组鸡盲肠粘膜上皮和肠腺上皮以及对照组和试验组的脾脏几乎不着色或着色很浅。试验结果提示，盲肠粘膜上皮和肠腺上皮的着色可能是诱导型N0S(iN0S)表达的结果，而由其产生的N0参与雏鸡球虫感染过程。     

口服BSA免疫对鸡盲肠扁桃体组织结构变化的影响

为了观察T细胞依赖性抗原对鸡盲肠扁桃体组织结构变化的影响,将雏鸡分为免疫组和对照组,每组50只,免疫组雏鸡定时进行空腹口服免疫1mL浓度为10mg/mL牛血清白蛋白(BSA),连续口服免疫6d。在最后一次免疫后的1、4、7、10、13、16、19、22、25、28d分别随机选取4只鸡,取其盲肠扁桃体。对照组也在各免疫组取材相同的日龄时取相同部位的组织,用HE染色法结合图像分析软件和数据统计软件对其进行观察。结果显示,免疫组鸡的盲肠扁桃体的肠绒毛长度、皱褶高度、肠腺深度﹑生发中心的数量和直径均比对照组鸡盲肠扁桃体的肠绒毛长、皱褶高﹑肠腺深﹑生发中心的数量多和直径大。说明口服免疫T细胞依赖性抗原对鸡盲肠扁桃体组织结构变化存在影响。     

柔嫩艾美尔球虫与肠炎沙门菌混合感染对雏鸡盲肠菌群的影响

为探讨柔嫩艾美尔球虫与肠炎沙门菌单独感染及混合感染对盲肠肠道菌群的影响,12日龄莱航鸡口服单独或混合感染柔嫩艾美尔球虫或肠炎沙门菌,18日龄通过16SrRNA Miseq高通量测序检测鸡盲肠菌群组成。结果表明,18日龄莱航鸡盲肠内主要菌群为厚壁菌门和拟杆菌门。与柔嫩艾美尔球虫或肠炎沙门菌单独感染相比,混合感染导致肠杆菌科明显增加,毛螺菌科明显减少;因此,柔嫩艾美尔球虫与肠炎沙门菌混合感染对盲肠肠道菌群的影响与两者单独感染明显不同。     

Kinetics of interleukin-2 production in chickens infected with Eimeria tenella

Interleukin (IL)-2 is a major cytokine of cell-mediated immunity (CMI). Because chickens infected with Eimeria, the causative agent of coccidiosis, develop a robust cell-mediated response against the parasite, we measured IL-2 concentrations in vivo and in vitro during the course of primary and secondary experimental Eimeria tenella infections. IL-2 levels in serum and culture supernatants of spleen lymphocytes stimulated with mitogen or E. tenella sporozoites were significantly increased on day 7 post-primary infection compared with control group. This peak in IL-2 coincided with the time of maximum intestinal lesions as measured by cecum lesion scores. By contrast, during secondary infection highest IL-2 concentrations preceded intestinal lesions by 5 days (day 2 versus day 7, respectively). These results confirmed that IL-2 production is augmented during experimental coccidiosis and suggested that cellular immunity elicited during an anamnestic response to parasite reinfection is mediated, at least in part, by IL-2.     

Circulating prolactin concentrations in chickens infected with Eimeria tenella

50000 oocysts of Eimeria tenella were inoculated into three-week-old cockerels and the effect of the infection (coccidiosis) on circulating concentrations of glucose, prolactin, sodium, potassium and haematocrit was determined. At day 5 of infection haematocrit was reduced and glucose was increased. From day 7 onwards prolactin concentration was elevated in infected birds compared with control and pair-fed birds. Plasma electrolytes were unchanged. It appears likely that loss of water resulting in osmotic changes during infection is the major reason for the observed changes in prolactin concentration in infected cockerels.     

Interleukin-2 production in SC and TK chickens infected with Eimeria tenella

SC and TK inbred chicken strains display differential protective immunity to coccidiosis, SC being more resistant and TK susceptible to disease. In this study, the association between interleukin (IL)-2 and disease phenotype was assessed by cytokine quantification in serum, duodenum, cecum, and spleen cell cultures of SC and TK chickens experimentally infected with Eimeria tenella. In general, after primary infection, SC and TK strains produced equivalent amounts of IL-2 in all sources examined. However, after secondary infection, SC animals displayed significantly greater IL-2 levels in serum and the duodenum compared with strain TK. IL-2 production after reinfection with Eimeria may be an important factor contributing to the genetic differences in coccidiosis between SC and TK chickens and provides a rational foundation for cytokine-based immunotherapeutic approaches to disease control strategies.     

Adhesion of bacteria to the cecal mucosal surface of conventional and germ-free chickens infected with Eimeria tenella.

When Salmonella typhimurium and Clostridium perfringens were tested in conventional chickens, larger numbers of S typhimurium and C perfringens adhered to Eimeria tenella-infected ceca than to uninfected ceca. In germ-free chickens, S typhimurium and C perfringens adhered to the E tenella-infected cecal mucosa more than to the uninfected cecal mucosa, but fewer Bacteroides vulgatus and Bifidobacterium thermophilum adhered to the E tenella-infected ceca than to the uninfected ceca. Many bacteria adhered to the lesions caused by E tenella as observed by scanning electron microscopy. On the basis of our findings, we suggest that infection with E tenella upsets the balance of competitive adherence of bacteria, allowing more colonization of S typhimurium and C perfringens.     

柔嫩艾美耳球虫表面抗原SAG2基因的克隆与表达

根据生物信息学预测的基因序列设计引物,应用RT-PCR方法从柔嫩艾美耳球虫第二代裂殖子总RNA扩增获得了鸡柔嫩艾美耳球虫表面抗原2(surface antigen 2,SAG2)基因序列,将其与pGEM-T easy载体连接后转化E.coliDH5α,筛选阳性克隆,以带有限制酶切位点的特异性引物用PCR方法扩增不含SAG2 N端信号肽序列的ORF序列后克隆至表达载体pET-32 a(+),构建了重组表达质粒pET-32 a(+)-SAG2,并将其转化至E.coliBL21(DE3)。经IPTG诱导,获得了SAG2重组抗原在大肠杆菌的高效表达,重组蛋白的表达量约占菌体总蛋白的35%,融合蛋白的分子量约为47 ku。菌体经超声处理后进行SDS-PAGE分析表明,表达蛋白以包涵体的形式存在。     

重组ChIFN-γ增强鸡球虫细胞免疫作用的研究

初步探讨了重组鸡γ干扰素(rChIFN-γ)对鸡体抗球虫细胞免疫的增强效果,分别给7日龄和14日龄雏鸡邓E.tenella孢子化卵囊免疫并同时肌肉注射500U rChIFN-γ后,于21日龄以同源E.tenella攻虫.试验结果显示:rChIFN-γ处理组的试验雏鸡脾脏和胸腺免疫器官指数显著高于免疫对照组(P＜0.05),在21日龄时的淋巴细胞转化水平(1.14)也显著高于卵囊免疫对照组(0.95)(P＜0.05);ABC染色结果表明rChIFN-γ可以显著增加试验雏鸡脾脏和盲肠的CD4 、CD8 T淋巴细胞数量;Griess(NO2-)结果表明:rChIFN-γ处理组在21日龄的NO2-含量(2.77ug/mL)显著高于免疫对照组(2.14 ug/mL)(P＜0.05);溶菌酶(LSZ)试验表明,21日龄时rChIFN-γ处理组的LSZ含量(22.80ug/mL)显著高于免疫对照组(19.79 ug/mL)(P＜0.05).结果提示rChIFN-γ具有抗球虫免疫的增强效果,能显著提高鸡体抗球虫保护性细胞免疫水平.     

Blackhead disease (Histomonas meleagridis) aggravated in broiler chickens by concurrent infection with cecal coccidiosis (Eimeria tenella).

The effect of concurrent cecal coccidiosis infections on severity of Histomonas meleagridis (blackhead disease) in chickens was investigated in a series of experiments. Cecal lesions from H. meleagridis were severe in all inoculated control groups and did not appear to be affected by the introduction of Eimeria tenella infection. However, the severity of liver lesions and number of birds positive for liver lesions of H. meleagridis increased significantly with the presence of E. tenella. The increase was similar when 10(3) or 10(4) oocysts of E. tenella were given and was the same when oocysts were given at the same time as H. meleagridis or 4 days prior. The liver lesions increased directly as doses of H. meleagridis increased from 7.5 x 10(3) cells to 30, 100, or 300 x 10(3) when E. tenella was given along with H. melelagridis but not when H. meleagridis was given alone. Administration of a live coccidiosis vaccine containing very low levels of E. tenella also gave a significant boost to liver lesions but at a much lower level than that observed with larger doses of E. tenella. The positive relationship between infections of cecal coccidiosis and H. meleagridis in chickens suggests that such dual exposure may contribute to increased clinical outbreaks of blackhead disease in chickens under field conditions.     

微球克对鸡柔嫩艾美耳球虫的预防效果试验

随着人们对健康和环境问题的日趋关注，使用化学药物造成的药物残留问题，引起了足够的重视。对于严重危害养禽业的鸡球虫病来说，目前，最主要的防治手段仍然是化学防治，但无药残、无环境污染的生物制品目前已逐渐应用到抗球虫上来。本试验拟在饲料中添加微生态添加剂微球克（益生菌群）以观察其对鸡柔艾美耳球虫病的防治效果。     

柔嫩艾美耳球虫（Eimeria tenella）感染鸡IgG生成细胞及血清IgG变化

利用免疫组化技术和Dot-ELISA，分别检测了雏鸡初次感染柔嫩艾美耳球虫（E.tenella)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以及不同抗体水平雏鸡的抗球虫能力。结果表明，（1）感染后2-3d，盲肠粘膜、脾脏、法氏囊、肓肠扁桃体中的IgG生成细胞即开始增殖，9-12d达峰值，随后开始下降，盲肠扁桃体中IgG生成细胞数在22d时仍高于对照组；（2）感染后6d即可在循环血液中检测到特异性IgG，18d达峰值，30d降至感染后7d时的水平；（3）特异性母源抗体IgG水平高的雏鸡，抗球虫能力高，二者呈明显的正相关。