鸡柔嫩艾美耳球虫发育相关基因的cDNA阵列检测及分析

为从分子水平上了解球虫发育的相关信息,本文以柔嫩艾美耳球虫的4个发育阶段(未孢子化卵囊、孢子化卵囊、子孢子和裂殖子)为试材,分别与本实验室制备的柔嫩艾美耳球虫cDNA阵列进行杂交。经过分析,在4个不同发育阶段共有484个基因特异表达,其中仅79个为功能注释基因,其余为功能未知基因。在子孢子阶段筛选到了以下重要基因:1.糖代谢相关基因如糖原磷酸化酶、葡萄糖酸透性酶和丙酮酸激酶;2.入侵宿主细胞相关基因如微线蛋白2(MIC2)、黏液素和类黏液素蛋白。信号转导相关基因如磷脂酰激醇4激酶在未孢子化卵囊阶段特异低丰度表达,但是根据4个发育阶段杂交数据的趋势线,这些基因在子孢子和裂殖子阶段应高丰度表达。本研究为研制新的抗球虫药物提供了实验依据。     

宿主日龄对艾美耳球虫 (Eimeria)早熟虫株感染力及雏鸡球虫免疫力的影响(英文)

用 10 0 0个柔嫩艾美耳球虫 (E .tenella)早熟株孢子化卵囊 ,10 0 0个毒害艾美耳球虫 (E .neca trix)早熟株孢子化卵囊和 5 0 0个巨型艾美耳球虫 (E .maxima)早熟株卵囊分别接种 1,7和 2 5日龄雏鸡 ,接种后 14天分别用相应的 2 0 0 ,2 0 0和 10 0个亲本毒株孢子化卵囊进行攻虫。初次接种和攻虫后分别检查卵囊总产量。结果表明 ,3种早熟虫株的卵囊都能够成功的感染 1日龄和 7日龄雏鸡。初次接种后雏鸡都产生了不同程度的球虫免疫力。本研究证实 ,艾美耳球虫早熟株卵囊能够诱导初生和幼龄雏鸡产生球虫免疫力     

宿主日龄对艾美耳球虫（Eimeria）早熟虫株感染力及雏鸡球虫免疫力的影响

用1000个柔嫩艾美耳球虫(E.tenella)早熟株孢子化卵囊,1000个毒害艾美耳球虫(E.necatrix)早熟株孢子化卵囊和500个巨型艾美耳球虫(E.maxima)早熟株卵囊分别接种1,7和25日龄雏鸡,接种后14天分别用相应的200,200和100个亲本毒株孢子化卵囊进行攻虫.初次接种和攻虫后分别检查卵囊总产量.结果表明,3种早熟虫株的卵囊都能够成功的感染1日龄和7日龄雏鸡.初次接种后雏鸡都产生了不同程度的球虫免疫力.本研究证实,艾美耳球虫早熟株卵囊能够诱导初生和幼龄雏鸡产生球虫免疫力.     

毒害艾美耳球虫江苏分离株抗原基因NA4的克隆与序列分析

采用直肠接种毒害艾美耳球虫（Eimeria necatrix）裂殖子方法对毒害艾美耳球虫卵囊进行纯化,用柔嫩艾美耳球虫子孢子表面抗原TA4基因特异性引物,以纯化E.necatrix孢子化卵囊总RNA为模板,RT-PCR方法克隆出了一段新基因序列。该基因全长747bp,共编码248个氨基酸,与国外Brothers等（1991）从E.necatrix孢子化卵囊表面得到的NA4基因同源性达99.7%,与柔嫩艾美耳球虫TA4（No.AJ586531.2）基因同源性达到91.4%,拥有与柔嫩艾美耳球虫TA4抗原基因相同序列的一段信号肽。从结果可得出,新克隆的基因为E.necatrix NA4基因。此基因已登录GenBank,登录号为EU523548。     

堆型艾美耳球虫cSZ1基因在减毒沙门氏菌的表达及免疫保护效果研究

用PCR方法扩增堆型艾美耳球虫广东株子孢子表面抗原基因cSZ1的阅读框架 (ORF) ,与质粒表达载体pYA3342连接后转化大肠杆菌E-coliX6 2 12 ,获得阳性重组质粒后将其电转化至减毒鼠伤寒沙门氏菌(Salmonellatyphimurium)X4 5 5 0。经IPTG诱导获得了cSZ1基因在减毒S typhimurium的高效表达 ,表达产物占菌体总蛋白的 9 2 % ,重组蛋白分子量约为 19kDa。将重组减毒S typhimurium分别以 10 8或 10 9CFU 鸡经口免疫 4日龄岭南黄肉鸡 ,免疫后两周血清中可检测到抗cSZ1的特异性IgG ,3周时抗体水平达到峰值 ;2 5日龄时可在肠道检测到特异性IgA的分泌。免疫后 3周 ,试验鸡分别经口攻击感染 5 0 0个E acervulina孢子化卵囊 ,结果显示免疫组与非免疫组有相似的排卵囊曲线 ;计数攻虫感染后第 3 5～ 9 5天卵囊总产量 ,2个免疫组分别能降低 2 5 . 1%和 4 6 . 4 %的卵囊产生。     

弓形虫RH株速殖子期表达型cDNA文库的构建及鉴定

为构建弓形虫速殖子期表达的基因的完整长度cDNA文库 ,用CF 11纤维素柱快速提纯速殖子 ,以盐酸异硫氰酸胍 (AGPC)一步法抽提总RNA ,oligo dT纤维素分离mRNA后 ,用ClonTech公司的SmartTMPCRcDNA文库构建试剂盒 ,构建了弓形虫RH株的表达型文库。获得 5× 10 6个独立克隆 ,重组率为 99% ,插入片段的平均长度约为 1kb。根据已知序列设计引物 ,从cDNA文库中扩增出编码棒状体蛋白 1(ROP1)的基因 (不含编码信号肽的序列 ) ,本文库可望用于弓形虫疫苗研究的候选基因的筛选。     

柔嫩艾美耳球虫（E.tenella）BJ株TA4基因的克隆的序列分析

对柔嫩艾美耳球虫（Ｅ．ｔｅｎｅｌｌａ）ＢＪ株ＴＡ４基因进行了克隆和测序分析。经纯化的Ｅ．ｔｅｎｅｌｌａＢＪ株７ｈ孢子化卵囊的总ＲＮＡ为模板，根据国外报道的序列设计一对引物，用ＲＴ－ＰＣＲ方法扩增出ＢＪ虫株的ＴＡ４基因。用常规基因克隆方法把ＢＪ株ＴＡ４基因插入ｐＧＥＭ－Ｔ克隆载体，选取正方向插入的一个阳性克隆进行酶切分析及插入片段的全序列测序分析。结果表明：该序列全长１２２７个核苷酸，有一个含２３０     

柔嫩艾美耳球虫(E.tenella)BJ株TA4基因的克隆和序列分析

对柔嫩艾美耳球虫(E.tenella)BJ株TA4基因进行了克隆和测序分析。以纯化的E.tenellaBJ株7h孢子化卵囊的总RNA为模板,根据国外报道的序列设计一对引物,用RT-PCR方法扩增出BJ虫株的TA4基因。用常规基因克隆方法把BJ株TA4基因插入pGEM-T克隆载体,选取正方向插入的一个阳性克隆进行酶切分析及插入片段的全序列测序分析。结果表明:该序列全长1227个核苷酸,有一个含230个密码子的开放性读框,可编码分子量约为25kDa的多肽。其后紧随533bp的3'端非编码序列。TA4-BJ与国外株TA4比较,同源性99%,突变的4个核苷酸中,2个为有义突变,使其推测氨基酸Asp变为Gly,Phe变为Leu     

三株柔嫩艾美耳球虫(Eimeria tenella)的繁殖力和免疫原性的比较研究

分别用柔嫩艾美耳球虫早熟弱毒株、鸡胚适应株和强毒株孢子化卵囊１０００个／只，给１０日龄无球虫雏鸡免疫接种，逐日检查粪便并进行克粪便卵囊（Ｏ．Ｐ．Ｇ．）计数，结果发现早熟弱毒株和鸡胚适应株的潜隐期均有不同程度缩短，繁殖力亦有不同程度的下降。同时进行了柔嫩艾美耳球虫不同株球虫的免疫原性比较：分别用三株卵囊免疫雏鸡，免疫剂量为１０００个／只，免疫后１４ｄ各用１０００个／只强毒株卵囊攻虫，逐日进行Ｏ．Ｐ．Ｇ．计数，发现各组免疫鸡攻虫后的卵囊产量均大大下降，其中以强毒株免疫攻虫组的排出卵囊量最低，仅为未免疫组的１．２３％，早熟株和鸡胚株免疫鸡攻虫的排出卵囊量相近，分别为未免疫组的７．３５％和７．２３％。由此可见，三株柔嫩艾美尔球虫均表现出良好的免疫原性。     

三株柔嫩艾美耳球虫（Eimeria tenella）的繁殖力和免疫原？…

分别用柔蒋美耳球虫早熟弱毒株、鸡胚适应株和强毒株孢子化卵囊１０００个／只，给１０日龄无早雏鸡免疫接种，逐日粪便并进行克粪便卵囊计数，结果发现产毒株和鸡胚适应株的潜隐期均有不同程度缩短，繁殖力亦有不同程度的下降，同时进行了柔嫩艾美耳球虫不同株球虫的免疫原性比较，分别用三株卵囊免疫雏鸡，免疫剂理为１０００个／只，免疫后１４ｄ各用１０００个／只强毒株卵囊攻虫，逐日进行Ｏ．Ｐ．Ｇ．计数，发现各组免疫鸡攻虫     

Eimeria infection of chicken embryos: the effect of known anticoccidial drugs against E. tenella and E. mivati

The activity of nine anticoccidial drugs was investigated against Eimeria infections of chicken embryos. With Eimeria tenella, three parameters were used to assess the antiparasitic effects (a) mortality (b) focal lesions on the chorioallantoic membrane (CAM) and (c) oocyst production. Anticoccidial drugs against E. mivati were evaluated by estimating oocyst production only. Only three of the anticoccidials tested were found to have high activity when measured by all of the above criteria. All of the drugs tested reduced infections caused by E. tenella and E. mivati. With f. tenella, inhibition of CAM focal lesions and oocyst production proved to be more sensitive parameters than mortality of infected embryos. The number of sporozoites in the inoculating dose was important, particularly in the oocyst production studies because excessively large numbers of sporozoites did not result in high yields of oocysts. The application of chicken embryo infections with Eimeria for the study of anticoccidial drugs is discussed.     

Characterization and immunoprotective properties of a monoclonal antibody against the major oocyst wall protein of Eimeria tenella.

The oocyst wall of Eimeria spp. consists of a 10-nm-thick outer lipid layer and a 90-mm-thick inner layer of glycoprotein which has been described previously to be composed of a single major protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and (125)I labelling of a oocyst wall fragments and of delipidated intact oocysts revealed a molecule of approximately 12 kDa as the major protein component of the oocyst wall of Eimeria tenella. An immunoglobulin M monoclonal antibody (c11B9F3) was produced against this 12-kDa oocyst wall protein sliced from a preparative SDS-polyacrylamide gel. Its reactivity by immunofluorescence against oocyst wall fragments and sporozoites or by immunoperoxidase assays of infected tissue sections was stage restricted to gametocytes and oocysts but pan-specific against all face of the oocyst wall. In chicks passively immunized with C11B9F3, oocyst output was significantly (P<0.01) reduced by 42 to 54% after homologous E. tenella infection and by 35% after heterologous Eimeria maxima infection compared with that of control groups. The results demonstrate the presence of a highly conserved, low-molecular-weight antigen on the oocyst wall and the gametocytes of Eimeria spp. which is a candidate for inclusion in a pan-specific, transmission-blocking vaccine against avian coccidiosis.     

Protein profiles of five avian Eimeria species.

SDS-PAGE fingerprint studies of oocyst antigens of five major Eimeria species including E. acervulina, E. maxima, E. necatrix, E. praecox and E. tenella demonstrated that their protein patterns are different, but there are some shared proteins between species and at least one protein band (45 kDa) was conserved among the five species. In Western blot studies, some species-specific as well as a few shared immunogenic bands were identified and chicken anti-E.maxima sera reacted with the conserved protein band in oocyst antigens of all these species.     

Eimeria tenella sporozoites and merozoites differentially express glycosylphosphatidylinositol-anchored variant surface proteins

Little is known about glycosylphosphatidylinositol (GPI)-linked surface proteins in the coccidian parasite Eimeria tenella. Examination of 28,550 EST sequences from the sporozoite and second merozoite developmental stages of the parasite led to the identification of 37 potential GPI-linked variant surface proteins, termed EtSAGs. Analysis of the complete nucleotide sequences of 23 EtSAG genes separated them into two multi-gene families. All the predicted EtSAG proteins (which vary in length from 228 to 271 residues) have an N-terminal hydrophobic signal peptide, a C-terminal hydrophobic GPI signal-anchor peptide and an extracellular domain organised around six cysteine residues, the positions of which are conserved within each family. Using specific antibodies against a small number of recombinant-expressed EtSAGs, the surface localisation and GPI-anchorage of members of both families was confirmed experimentally. Expression of EtSAGs is differentially regulated between the oocyst/sporozoite and second generation merozoite stages, with only one expressed specifically in the sporozoite, a small number expressed in both stages and the majority expressed specifically in the second generation merozoite. Preliminary data support a model in which multiple variant surface antigens are co-expressed on individual parasites, rather than a model of antigenic switching. The biological role(s) of EtSAGs and the effect(s) that expression of a complex repertoire of variant surface antigens by the second generation merozoite has on host adapted immunity are unknown.     

Detection of Eimeria tenella antibodies by the serum dye test

The serum dye test by Sabin-Feldman used in the toxoplasmosis serology was adapted to the Eimeria tenella system. The antibodies detected by this specific test reflect a protective immune response. All animals immunized with virulent and attenuated Eimeria tenella oocyst antigens demonstrated serologically immune reactions. Antigen reactivities over 50% correlate with the grade of protection of animals in challenge test.     

Cross-reactivity among antisera raised against five avian Eimeria species in the natural host and in rabbits

Water- and SDS-soluble antigens were prepared from purified sporulated oocysts of Eimeria acervulina, E. maxima, E. necatrix, E. praecox and E. tenella. Reactivity of . chicken hyperimmune anti-Eimeria sera, rabbit anti-oocyst and rabbit anti-sporozoite sera with the homologous and heterologous oocyst antigens were determined and cross-reactivities were expressed as a percentage of those homologous sera. The results demonstrated that the antisera from chickens infected naturally with Eimeria species differed in their reactivities from those of the rabbit antisera. Occurrence of a high level cross-reactivity among the chicken antisera may suggest that the development of parasites inside the host cells, or the production of substances during the life-cycle, affect the extent of immune responses and that most non-protective antibodies are cross-reactive.     

Cross-protection against four species of chicken coccidia with a single recombinant antigen

A cDNA clone, SO7', from an Eimeria tenella cDNA library was inserted into the high-expression vector pJC264 and was expressed in Escherichia coli as a fusion protein, CheY-SO7', with a molecular mass of approximately 36 kDa. By using the purified recombinant antigen to immunize young chicks, it was demonstrated that a single dose, without adjuvant, not only protected against severe coccidiosis induced by infection with E. tenella but also protected chicks challenged with the heterologous species Eimeria acervulina, E. maxima, and E. necatrix. By using rabbit antiserum raised against recombinant CheY-SO7', Western blot (immunoblot) analysis of sporulated oocysts of all seven major species of chicken coccidia showed that all species tested contained proteins characteristic of the B class of antigens, of which CheY-SO7' is representative. It seems likely that a single B antigen could protect chickens against severe coccidiosis caused by infection with any of these Eimeria species. Although chicks exposed to prolonged, natural infection develop antibodies to B antigen, active immunization of young chicks with a protective dose of CheY-SO7' does not elicit a humoral antibody response, suggesting that the partial protection results from cell-mediated effector mechanisms. In addition, the cross-protective nature of the immunity indicates that the response to B antigen is different from that induced by natural infection, which elicits a species-specific immunity. To date, the protection induced by B antigen immunization, although remarkable for a single recombinant protein, is not sufficient to compete with prophylactic chemotherapy.