E.tenella感染雏鸡特异性抗体的动态变化和母源免疫的研究

本实验用斑点酶联免疫试验 (Dot ELISA)检测了E .tenella感染雏鸡特异性抗体、免疫母鸡卵黄和后代血清中特异性母源抗体的动态变化及不同日龄雏鸡的抗球虫水平。结果表明 :(1)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 18天达到峰值 ,第 30天降至感染后第 7天时的水平。 (2 )母鸡二次免疫后第 5天卵黄中即有高水平的母源抗体 ,免疫母鸡后代 1日龄时母源抗体滴度高达 9.83,与未免疫对照组差异极显著 (P <0 .0 1) ,二者都随着日龄增长降低 ,但免疫组下降幅度很小 ,2 9日龄时仍和对照组 18日龄水平相当。 (3)免疫母鸡后代对再感染的抵抗力显著增强 ,卵囊减少率高达 93%,抗球虫指数和相对增重率都与对照组差异极显著 (P <0 .0 1) ,雏鸡的抗球虫水平随着日龄增长逐渐降低 ;母源抗体、抗球虫指数和相对增重率三者之间两两呈现明显的正相关 ,而这三者又都与卵囊产量呈明显的负相关。     

柔嫩艾美耳球虫安徽肥西(AHFX)株的分离与鉴定

目的 分离并鉴定安徽肥西病鸡盲肠内的柔嫩艾美耳球虫.方法 通过雏鸡进行单卵囊接种与增殖试验,对所获卵囊用形态学方法进行初步鉴定;运用PCR方法扩增该分离株的ITS-1基因序列,并与相关序列进行比对、构建系统进化树.结果 该球虫分离株孢子化卵囊的平均大小为21.1 μm ×18.0 μm,卵形指数为1.17,潜隐期为140 h;其ITS-1基因序列与柔嫩艾美耳球虫(Eimeria tenella)GQ856310相似性达98.2％,二者的亲缘关系非常接近.结论 该球虫分离株为柔嫩艾美耳球虫(E.tenella),暂命名为柔嫩艾美耳球虫安徽肥西(AHFX)株.     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

鸡球虫18S rRNA基因序列的测定与分析

为了利用18S rRNA基因进行鸡球虫系统进化分析,对巨型艾美耳球虫(Eimeria maxima)、柔嫩艾美耳球虫(E.tenella)、堆形艾美耳球虫(E.acervulina)3种共8个不同来源的虫株,分别提取总DNA进行18S rRNA基因的扩增和测序;将得到的序列登录GenBank进行同源性和趋异性分析,并结合GenBank中其它原虫的18S rRNA基因序列构建进化树.结果显示扩增获得8株鸡球虫18S rRNA基因长度为1746～1756 bp,序列比对显示同种不同株间的同源性大于不同种间的同源性,其中3株E.maxima株间同源性在98.7%～99.3%之间,4株E.tenella株间同源性在99.7%～99.9%之间;不同种间同源性为96.5%～98.1%,其中E.maxima与E.tenclla的遗传距离最大,为0.038;E.maxima与E.acervulina的遗传距离最小,为0.021.顶复器门9个不同属所构建的进化树结果显示,E.imeria和等孢属(Isospora)聚为一支,说明亲缘关系比较近.与GertBank中其它5株不同鸡球虫的18S rRNA基因共同构建的进化树显示,3株E.maxima聚为一支,与E.brunetti、E.mitis、E.mivati、E.praecox和E.acervulina聚为一大分支;4株E.tenella与1株E.necatrix共同形成一个分支,说明E.tenella与E.necattix的亲缘关系最近.本研究证实了在鸡球虫系统进化研究中,18S rRNA基因不仅可以区分不同种,而且有可能成为区分同种不同株的理想靶基因.     

柔嫩艾美耳球虫重组鸡痘病毒的构建与免疫保护研究

以鸡柔嫩艾美耳球虫(E.tenella)杂交株F2的RNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术扩增了杂交株F2的Rhomboid蛋白家族相关基因,将PCR产物克隆至pMD18-T载体中,构建出克隆质粒pMD-Rhomboid.以KpnⅠ、PstⅠ双酶切重组质粒pMD-Rhomboid和鸡痘病毒载体质粒pUTA2,并将纯化的Rhomboid基因亚克隆至鸡痘病毒载体pUTA2复合启动子下游,构建出真核表达重组质粒pUTA-Rhomboid.采用脂质体转染技术,将该质粒转染FPV282E4株感染的鸡胚成纤维细胞(CEF)中,通过BrdU药物加压筛选,并通过RT-PCR和蛋白质印迹等方法检测,筛选出一株球虫鸡痘重组病毒rFPV-Rhomboid.进一步经CEF扩增病毒后,免疫雏鸡,监测免疫指标.结果表明:重组病毒接种鸡外周血中的CD4 、CD8 含量显著高于非免疫对照组(P<0.05);与对照组相比,重组病毒对鸡的增重效果差异显著(P<0.05),对E.tenella的攻击具有一定的保护作用,显示出较好的应用前景.     

抗生殖器疱疹病毒卵黄免疫球蛋白(IgY)的研究

检测了鸡卵黄中抗生殖器疱疹病毒(HSV-2)抗体的产量、纯度、来源及稳定性。采用生殖器疱疹病毒(HSV-2)作为抗原免疫广州黄村鸡。通过改良水稀释法提取卵黄中的IgY。双紫外光波长测定抗体含量,SDS-PAGE电泳检测抗体纯度。Western blot免疫印迹法测定该抗体来源。ELISA检测IgY对温度、酸碱度的稳定性。结果,蛋黄液中抗体质量浓度13.6g.L-1,抗体纯度达96.2%。免疫印迹证明IgY与鸡血清中的IgG具有相同的分子量和抗原性。IgY具有良好的热稳定性,对酸碱具有一定的耐受力。WD水稀释法能得到高产量、高纯度的特异性IgY,而且有良好的生物学活性。     

高原姬鼠一新艾美球虫记述(顶复合门,艾美球虫科)

粪检云南省昆明市太平镇捕获的31只高原姬鼠Apodemus chevrieri,在9只高原姬鼠中共发现了2种艾美球虫,自然感染率为29%,其中1种被命名为新种:齐氏艾美球虫Eimeria chevrieri sp.nov.。齐氏艾美球虫孢子化的卵囊椭圆形,大小为21·2μm×16·3μm(22·2~19·7μm×17·3~15·6μm)。卵囊壁光滑,单层,厚约1·0μm;无胚孔和卵囊余体。有1~2个极体,极体形状不规则,直径1·0~2·0μm。卵圆形的孢子囊大小为11·2μm×7·5μm(12·3~10·3μm×8·3~6·8μm),有孢子囊余体,且主要分布在2个子孢子的中间,斯氏体扁豆形,大小为1·0μm×0·3μm,亚斯氏体亚球形,大小为2·5μm×1·5μm。子孢子在孢子囊里面头尾相接,在其宽端有1个明显的反光体,直径约3μm。模式标本保存在云南大学标本馆。齐氏艾美球虫,新种Eimeria chevrieri sp·nov.(图1~2,4)寄生于姬鼠属,具有椭圆形的卵囊,卵囊壁具有光滑、无色、单层特点的艾美球虫有3种:E.gomurica Musaev and Veisov,1963,E.kaunensis Arnastauskiene et al.,1978和E.gandobica Musaev and Veisov,1965。E.gomurica、E.kaunensis与本种的主要区别是卵囊较小且有卵囊余体;E.gandobica与本种的主要区别是卵囊呈宽椭圆形,卵囊和孢子囊较小(19·0μm×16·8μmvs·21·2μm×16·3μm;8·0μm×5·0μmvs·11·2μm×7·5μm),孢子囊余体少,无反光体和亚斯氏体。寄生于姬鼠属,具有椭圆形的卵囊,卵囊壁具有光滑、2层特点的艾美球虫有3种:E.alorani Hurková et al.,2005、E.apodemi Pell啨rdy1974和E.zaurica Musaev and Veisov,1965。E.zaurica与本种的主要区别是具有卵囊余体;E.alorani的卵囊和孢子囊比本种大的多(26·9μm×19·3μmvs.21·2μm×16·3μm),且其孢子囊余体形成一个直径约4~5μm的球状结构;E.apodemi的孢子化卵囊呈宽椭圆形,经常不对称,无极体和斯氏体。该种区别其它种的关键特征是:1)孢子化卵囊椭圆形,卵囊壁光滑、单层、无色;2)有极体;3)孢子囊有亚斯氏体。模式标本,含孢子化卵囊的齐氏姬鼠粪便保存在云南大学生物系,标本编号:2006047;齐氏姬鼠标本保存于云南大学标本馆,标本编号20060081。词源:种名源自宿主种名。     

抗胱抑素C鸡卵黄IgY抗体的制备及鉴定

目的:制备高效价、高特异性的抗人胱抑素 C 鸡卵黄 IgY 抗体,并对其基本特性进行分析和鉴定.方法:以人胱抑素 C 为抗免疫产蛋的罗曼鸡,采用水稀释-盐析法提取及纯化 IgY 抗体,采用蛋白质定量、SDS-PAGE、West?ern 印迹和 ELISA 法对 IgY 抗体进行分析和鉴定.结果:免疫后14 d 即可从鸡冠血中检测出抗胱抑素 C 的特异性抗体,抗体效价在28 d 达最高峰(1∶32000),并可维持2个月以上;收集高效价时的免疫鸡蛋,制备鸡卵黄抗体 IgY;还性 SDS-PAGE 显示抗体 IgY 为相对分子质量分别为65×103和21×103的2条带,抗体纯度可达92%,得率为每个鸡蛋36.5 mg,抗体检出敏感度为15.63 ng/mL;Western 印迹证明该抗体具有高度特异性.结论:制备了抗胱抑素 C 的高效价、高特异性 IgY 抗体.     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫（Eimeria tenella）孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交（SSH）技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示：从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

60Coγ射线对高免卵黄液中EDS-76病毒灭活的研究

用不同剂量、剂量率的^60Coγ射线对卵黄液中的EDS-76病毒进行辐照,研究了其对病毒的灭活效果和对卵黄抗体(IgY)的影响。结果表明,^60Coγ射线辐照,可以灭活高免卵黄液中的EDS-76病毒,且随辐照剂量、剂量率的增大,灭活率也增高;EDS-76病毒在卵黄液中的D10值为0．57kGy～0．60kGy;6．0kGy的辐照剂量,可以将高免卵黄液中的EDS-76病毒完全灭活,且不影响卵黄抗体效价,该卵黄抗体稳定性好、耐酸、耐热、耐蛋白酶消化。     

Flotillin-1 localization on sporozoites oF Eimeria tenella

In an attempt to identify parasite surface components involved in the interaction with the host cell, the present research focuses on the rafts of Eimeria tenella that might be involved in the host cell invasion process. To that end, this study was undertaken to investigate the expression of flotillin-1, which is an important component and marker of lipid rafts at the plasma membrane of sporozoites of E. tenella. The expression of this plasma membrane protein was identified by an antibody that specifically reacts with flotillin- and was studied by electron microscopy. Flotillin-1 was found to occur in patches on the surface of E. tenella sporozoites. Immunoblot analysis of the total proteins of the sporozoites showed only 1 band of approximately 48 kDa. This indicates that the antibody exclusively recognized the molecules of flotillin-1 expressed on the surface of E. tenella sporozoites. The presence of flotillin-1 on the cellular membrane of sporozoites predominantly at the apical tip suggests that flotillin-1 belongs to the invasion machinery of E. tenella.     

Ultrastructural localization of antigenic sites on sporozoites, sporocysts, and oocysts of Eimeria acervulina and Eimeria tenella by monoclonal IgG and IgM antibodies

Immunoelectron microscopy was used to study the localization of monoclonal IgG (13.9 and 15.84) and IgM (10.84) antibodies generated against Eimeria tenella sporozoites on sporozoites, sporocysts, and oocysts of Eimeria acervulina and E. tenella. A uniform layer of ferritin was present on sporozoites of E. tenella fixed chemically before the addition of 10.84, 13.90, or 15.84 (called prefixed), whereas postfixed (fixed chemically after exposure to monoclonal antibody) sporozoites lacked ferritin, indicating that the latter had capped immune complexes. Patches of ferritin were present on prefixed and postfixed sporozoites of E. acervulina exposed to 15.84, indicating that immune complexes containing 15.84 were not capped. Sporocysts of E. tenella exposed to 10.84 had a uniform layer of ferritin on their outer surface; ferritin was localized in patches on those exposed to 13.90 or 15.84. In E. acervulina sporocysts exposed to 15.84, ferritin was widely scattered on the outer surface but formed a uniform layer on the inner surface of the sporocyst wall. Patches of ferritin occurred on the inner layer of the oocyst walls of E. tenella and E. acervulina exposed to 10.84, 13.90, or 15.84. These findings indicate the shared antigen detected by 15.84 differed in relative amount, spatial distribution, and structural location in sporozoites and sporocysts of E. acervulina and E. tenella.     

Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, E. maxima, E. necatrix, and E. tenella.

Merozoites of Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria tenella were compared by gel electrophoresis, western-blotting with chicken antiserum, indirect fluorescent antibody reactions, and antiserum neutralization. Merozoites from the 4 species had dissimilar patterns of proteins and antigens in soluble and membrane fractions. Coomassie blue staining of SDS-PAGE gels revealed 16-22 protein bands depending on the species of merozoite but only 3 bands per species in the membrane fractions. Homologous and heterologous antisera recognized 5-12 soluble fraction bands and 3-7 membrane fraction bands on immunoperoxidase-stained western blots, depending on the species. When antisera from infected chickens were used in an indirect fluorescent antibody reaction, the merozoites of E. tenella and E. necatrix had a strong reaction with homologous and heterologous antisera. Merozoites of E. acervulina and E. maxima reacted with homologous antisera but had a weak or no reaction with heterologous antisera. Chicken antiserum against E. tenella had no effect on the viability of E. tenella merozoites when they were inoculated into chicken embryos.     

Production and partial characterization of monoclonal antibodies specific for the gamonts of Eimeria tenella.

Thirteen hybridomas secreting monoclonal antibodies (Mabs) specific for the sexual stages (gamonts) of Eimeria tenella were produced by fusing spleen cells of gamont-immunized RBF/Dn mice with FOX-NY myeloma cells. A Mab subisotype profile revealed 1 IgG2a and 12 IgG1. All Mabs demonstrated a similar binding pattern when incubated with parasitic gamonts as determined by the indirect fluorescent antibody test. Ascitic fluid containing Mab (GD9 (IgG1) was produced and used to immunize chicks passively per os. There was a 34% decrease (P less than 0.05) in oocyst output from immunized chicks when compared to control chicks. Passively immunized chicks also had reduced cecal lesion scores when compared to control chicks. These results suggest that Mab GD9 partially inhibited the fertilization process of E. tenella.     

Eimeria tenella: use of a monoclonal antibody in determining the intracellular fate of the refractile body organelles and the effect on in vitro development

A monoclonal antibody, which recognizes the refractile body of Eimeria sporozoites, was used to study the developmental fate of this organelle during asexual development of E. tenella and to determine the effect of this monoclonal antibody on in vitro development of the parasite. Through use of immunofluorescent antibody and gold-labeling techniques at the light and electron microscopy level, the refractile body at 48 to 96 hr postinoculation was found to separate into 6 to 10 small globules, then diffuse throughout the schizont cytoplasm, and eventually reconcentrate as a small dot of material in each of the mature first-generation merozoites. The schizont did not develop to maturity if diffusion of the refractile body did not occur. The refractile body material was quickly lost as the merozoite left the schizont and invaded new cells and was not detected in any later developmental stages. The in vitro development of first- and second-generation schizonts of E. tenella was greatly inhibited (up to 100%) with exposure to the monoclonal antibody. There was an increase in the number of schizonts with nondispersed refractile body in the monoclonal antibody-treated cells when compared to the untreated controls, and the few mature schizonts seen had up to a 50-fold decrease in the number of merozoites. Immunofluorescent antibody labeling of the refractile body of intracellular sporozoites and schizonts treated in vitro with the monoclonal antibody for 24-96 hr postinoculation indicated that the antibody had crossed the host cell and parasite plasma membrane during incubation.     

Development of Eimeria tenella in MDBK cell culture with a note on enhancing effect of preincubation with chicken spleen cells

Eimeria tenella, an intracellular protozoan parasite infecting the epithelial cells of the ceca of chickens, causes severe diarrhea and bleeding that can lead its host to death. It is of interest that E. tenella first penetrate into the mucosal intraepithelial lymphocytes (IEL) before they parasitize crypt or villous epithelial cells. This in vitro study was undertaken to know whether the penetration of E. tenella into such a lymphoid cell is a beneficial step for the parasite survival and development. Three sequential experiments were performed. First, the in vitro established bovine kidney cell line, MDBK cells, were evaluated for use as host cells for E. tenella, through morphological observation. Second, the degree of parasite development and multiplication in MDBK cells was quantitatively assayed using radioisotope-labelled uracil (3H-uracil). Third, the E. tenella sporozoites viability was assayed after preincubation of them with chicken spleen cells. E. tenella o?cysts obtained from the ceca of the infected chickens were used for the source of the sporozoites. Spleen cells (E) obtained from normal chickens (FP strain) were preincubated with the sporozoites (T) at the E:T ratio of 100:1, 50:1 or 25:1 for 4 or 12 hours, and then the mixture was inoculated into the MDBK cell monolayer. Morphologically the infected MDBK cells revealed active schizogonic cycle of E. tenella in 3-4 days, which was characterized by the appearance of trophozoites, and immature and mature schizonts containing merozoites. The 3H-uracil uptake by E. tenella increased gradually in the MDBK cells, which made a plateau after 48-60 hours, and decreased thereafter. The uptake amount of 3H-uracil depended not only upon the inoculum size of the sporozoites but also on the degree of time delay (preincubation; sporozoites only) from excystation to inoculation into MDBK cells. The 3H-uracil uptake became lower as the preincubation time was prolonged. In comparison, after preincubation of sporozoites with spleen cells for 4 or 12 hours, the 3H-uracil uptake was significantly increased compared with that of control group. From the results, it was inferred that, although the penetration of E. tenella sporozoites into the lymphoid cells such as IEL is not an essential step, it should be at least a beneficial one for the survival and development of sporozoites in the chicken intestine.     

Partial protection against Eimeria acervulina and Eimeria tenella induced by synthetic peptide vaccine

Coccidiosis is a major parasitic disease of poultry industry and an ideal vaccine should induce long-lasting cross-species protective immunity. Broiler chickens (Cobb 500) were inoculated with single, double or triple injections of a synthetic peptide (derived from sequences of Eimeria acervulina and Eimeria tenella antigens) homogenized in Freund's complete and incomplete adjuvants. The immune responses to the vaccine were assessed by evaluation of antibody and lymphocyte proliferation responses, and the degree of resistance of vaccinated chickens to challenge with sporulated oocysts of E. acervulina or E. tenella determined by comparison of their oocyst output with those of control chickens. The results indicated that the synthetic peptide vaccine induced a high level of antibody and cellular responses associated with partial cross-species protection against challenge with sporulated oocysts of E. acervulina or E. tenella.     

Cell-mediated immunity to Eimeria in the fowl: the absence of cross-species protection is not due to the lack of cross-reactive T cells.

Immunity to Eimeria species in the fowl has been shown to be species specific and it has been proposed that the lack of cross-protection between the species of Eimeria is due to the absence of T cells which recognize antigens from a heterologous species. When this hypothesis was tested the results showed that antigens from E. tenella elicited a strong specific response by cells from birds immune to E. tenella. In contrast cells from birds which were immune to E. acervulina responded to a similar magnitude to both E. tenella and E. acervulina antigens. This indicates that the lack of cross-protection is not due to the lack of cross-reactive T cells.