Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina

Vaccination with proteins from gametocytes of Eimeria maxima protects chickens, via transfer of maternal antibodies, against infection with several species of Eimeria. Antibodies to E. maxima gametocyte proteins recognise proteins in the wall forming bodies of macrogametocytes and oocyst walls of E. maxima, Eimeria tenella and Eimeria acervulina. Homologous genes for two major gametocyte proteins - GAM56 and GAM82 - were found in E. maxima, E. tenella and E. acervulina. Alignment of the predicted protein sequences of these genes reveals that, as well as sharing regions of tyrosine richness, strong homology exists in their amino-terminal regions, where protective antibodies bind. This study confirms the conservation of the roles of GAM56 and GAM82 in oocyst wall formation and shows that antibodies to gametocyte antigens of E. maxima cross-react with homologous proteins in other species, helping to explain cross-species maternal immunity.     

Stable transfection of Eimeria tenella: constitutive expression of the YFP-YFP molecule throughout the life cycle

The obligate intracellular apicomplexan parasite Eimeria tenella, one of seven species of Eimeria that infect chickens, elicits protective cell-mediated immunity against challenge infection. For this reason, recombinant E. tenella parasites could be utilised as an effective vaccine vehicle for expressing foreign antigens and inducing immunity against heterologous intracellular microbes. A stable line of E. tenella expressing foreign genes is a prerequisite, and in this work an in vivo stable transfection system has been developed for this parasite using restriction enzyme-mediated integration (REMI). Two transgenic populations of E. tenella have been obtained that express YFP-YFP constitutively throughout the parasite life cycle. Southern blotting and plasmid rescue analyses show that the introduced exogenous DNA was integrated at random into the parasite genome. Although the life cycle of the transgenic populations was delayed by at least 12 h and the output of oocysts was reduced 4-fold relative to the parental BJ strain of E. tenella, the transgenic parasites were sufficiently immunogenic to protect chickens against challenge with either transgenic or parental parasites. These results are encouraging for the development of transgenic E. tenella as a vaccine vector and for more detailed investigation of the biology of the genus Eimeria.     

Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens

Recently, the availability of protocols supporting genetic complementation of Eimeria has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors, expressing and delivering heterologous proteins. Complementation with sequences encoding immunoprotective antigens from other Eimeria spp. offers an opportunity to reduce the complexity of species/strains in anticoccidial vaccines. Herein, we characterise and evaluate EtAMA1 and EtAMA2, two members of the apical membrane antigen (AMA) family of parasite surface proteins from Eimeria tenella. Both proteins are stage-regulated, and the sporozoite-specific EtAMA1 is effective at inducing partial protection against homologous challenge with E. tenella when used as a recombinant protein vaccine, whereas the merozoite-specific EtAMA2 is not. In order to test the ability of transgenic parasites to confer heterologous protection, E. tenella parasites were complemented with EmAMA1, the sporozoite-specific orthologue of EtAMA1 from E. maxima, coupled with different delivery signals to modify its trafficking and improve antigen exposure to the host immune system. Vaccination of chickens using these transgenic parasites conferred partial protection against E. maxima challenge, with levels of efficacy comparable to those obtained using recombinant protein or DNA vaccines. In the present work we provide evidence for the first known time of the ability of transgenic Eimeria to induce cross protection against different Eimeria spp. Genetically complemented Eimeria provide a powerful tool to streamline the complex multi-valent anticoccidial vaccine formulations that are currently available in the market by generating parasite lines expressing vaccine targets from multiple eimerian species.     

Novel plasmid-based genetic tools for the study of promoters and terminators in Streptococcus pneumoniae and Enterococcus faecalis

Promoter-probe and terminator-probe plasmid vectors make possible to rapidly examine whether particular sequences function as promoter or terminator signals in various genetic backgrounds and under diverse environmental stimuli. At present, such plasmid-based genetic tools are very scarce in the Gram-positive pathogenic bacteria Streptococcus pneumoniae and Enterococcus faecalis. Hence, we developed novel promoter-probe and terminator-probe vectors based on the Streptococcus agalactiae pMV158 plasmid, which replicates autonomously in numerous Gram-positive bacteria. As reporter gene, a gfp allele encoding a variant of the green fluorescent protein was used. These genetic tools were shown to be suitable to assess the activity of promoters and terminators (both homologous and heterologous) in S. pneumoniae and E. faecalis. In addition, the promoter-probe vector was shown to be a valuable tool for the analysis of regulated promoters in vivo, such as the promoter of the pneumococcal fuculose kinase gene. These new plasmid vectors will be very useful for the experimental verification of predicted promoter and terminator sequences, as well as for the construction of new inducible-expression vectors. Given the promiscuity exhibited by the pMV158 replicon, these vectors could be used in a variety of Gram-positive bacteria.     

An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin

Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freund’s Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens.     

Cross immunity of DNA vaccine pVAX1-cSZ2-IL-2 to Eimeria tenella, E. necatrix and E. maxima

The study describes cross protection experiments with chimeric DNA vaccine pVAX1-cSZ2-IL-2 to determine its efficacy against four important Eimeria species. Seven-day-old chickens were randomly divided into nine groups; group 1 negative control, groups 2, 3, 4, 5 positive controls; and groups 6, 7, 8 and 9 experimental groups. On days 7 and 14, groups 1-5 were injected with TE buffer, and groups 6-9 with the vaccine. At 21 days of age, all chickens were inoculated with 5 × 10⁴ sporulated oocysts except for the negative control. Groups 2 and 6 were inoculated with Eimeria tenella, groups 3 and 7 with Eimerianecatrix, groups 4 and 8 with Eimeria acervulina and groups 5 and 9 with Eimeria maxima. Seven days later, all chickens were weighed and slaughtered to obtain intestinal samples. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, lesion score, body-weight gain and anti-coccidial index. The results indicated that the recombinant plasmid can induce host immune responses by alleviating intestinal lesions, body weight loss and oocyst ratio and imparting good protection against E. tenella and E.acervulina, medium protection against E. necatrix but little effect against E. maxima. It is concluded that the conserved antigen can provide cross protection and should be explored further.     

Developing an in vitro method for Eimeria tenella attachment to its preferred and non-preferred intestinal sites

A frozen section method utilising chicken intestinal tissue was developed to study the Eimeria tenella attachment ex vivo. In order to examine Eimeria-epithelial cell attachment, 10⁵E. tenella sporozoites were incubated with each caecal frozen section (6, 10 and 14 μm) for 1 h in 5% CO₂ incubator at 41 °C. E. tenella sporozoites attached successfully to enterocytes in 14 μm thick of caecal sections. Sporozoite attachment to caecal sections was shown to be dependent on the number of parasites added. To evaluate the method, E. tenella sporozoites were incubated to its preferred (caecum) and non-preferred (duodenum and jejunum) intestinal sites. The number of sporozoites attached to the caecal enterocytes was significantly greater (P < 0.0001) in comparison with the limited number of sporozoites attached to enterocytes of non-preferred intestinal sites. This method was shown to be able to reveal differences in binding capability and allows for comparison of intestinal site attachment.     

Construction of two vectors for gene expression in Trichoderma reesei

We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei. The two newly constructed vectors can be efficiently transformed into T. reesei with Agrobacterium-mediated transformation. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. As a result, pWEF32 easily undergoes homologous recombination. On the other hand, pWEF31 undergoes random recombination. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30. Additionally, we measured the exo-1,4-β-glucanase activity of the recombinant cells. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. reesei. It also provides a method for recombination at a specific chromosomal location. Finally, both vectors will be useful for the large-scale gene expression industry.     

A survey of genes in Eimeria tenella merozoites by EST sequencing

A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.     

Improved techniques for endogenous epitope tagging and gene deletion in Toxoplasma gondii

Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0 kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii.     

Expression of multi-functional cellulase gene mfc in Coprinus cinereus under control of different basidiomycete promoters

Multi-functional cellulase gene mfc was expressed in Coprinus cinereus under naturally non-inductive conditions using three heterologous promoters. Endo-β-1,4-glucanase expression was achieved in solid and liquid media with promoter sequences from the Lentinula edodesgpd gene, the Flammulina velutipes gpd gene and the Volvariella volvaceagpd gene. As measured by enzyme activity in liquid cultures, a 613-bp gpd promoter fragment from L. edodes was most efficient, followed by a 752-bp gpd fragment from F. velutipes. The V. volvacea gpd promoter sequence was less active, in comparison. Irrespective of the promoter used, enzymatic activities increase 34-fold for highly active transformants and 29-fold for less active one by using cellulase-inducing medium. The highest activities of endo-β-1,4-glucanase (34.234 U/ml) and endo-β-1,4-xylanase (263.695 U/ml) were reached by using the L. edodesgpd promoter.     

Eimeria trichosuri: Phylogenetic position of a marsupial coccidium, based on 18S rDNA sequences

Phylogenetic analysis of the genus Eimeria suggests that parasite and host have coevolved over broad evolutionary timescales. Here we extend this analysis by determining the 18S rDNA gene sequence of the marsupial coccidium, Eimeria trichosuri, and assessing its phylogenetic position relative to Eimeria from birds, reptiles and placental mammals. This analysis placed E. trichosuri clones in a clade that diverged before the major clade comprising species from placental mammals. The position of E.trichosuri is consistent with host phylogeny where marsupials represent an ancient evolutionary line that predates the placental mammal line.     

Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 10⁴ homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4⁺ and CD8⁺ lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4⁺ and CD8⁺ T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant non-antibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens.     

高榕榕果内Eupristina属两种榕小蜂的遗传进化关系

对生长在福州地区的高榕进行长期追踪观察,发现高榕榕果内仅生活着Eupristina altissima和Eupristina sp.榕小蜂,前者为高榕的传粉小蜂,后者无传粉行为,两者雌蜂之间在体色、触角、花粉袋和花粉刷等部位存在细微的差异,而两者雄蜂之间无形态差异。通过克隆福建地区5个样地的高榕榕果内收集到的E. altissima和Eupristina sp.榕小蜂,以及细叶榕的传粉小蜂Eupristina verticillata(外群)的Cytb及COI基因,并进行碱基组成及遗传距离分析,用邻接法构建系统发育树,分析两榕小蜂群体之间的遗传进化关系,结果显示:(1)榕小蜂COI及Cytb序列碱基组成中A+T的含量(Cytb序列中A+T=75.3%,COI序列中A+T=75.5%)显著高于G+C,符合膜翅目昆虫线粒体基因碱基组成特征。(2)对两群体小蜂进行遗传距离分析显示,Cytb序列中E. altissima和Eupristina sp. 群体内各样本之间的平均遗传距离分别为0.0092和0.0030,而E. altissima与Eupristina sp. 群体间的平均距离为0.1588;COI序列中E. altissima 与Eupristina sp. 群体内各样本之间的平均遗传距离分别为0.0065和0.0205,而二者群体间的平均遗传距离为0.1043,表明两者群体间的遗传距离明显大于各自群体内各样本间的遗传距离。统计GenBank中下载的6个属34种榕小蜂Cytb序列的种间遗传距离为0.0811-0.1723,6个属28种榕小蜂COI序列的种间遗传距离为0.0939-0.1986。由此认为E. altissima和Eupristina sp.之间的遗传距离差异已经达到了种间水平,即E. altissima与Eupristina sp.为两个不同的种。(3)在形态上,两种小蜂的雌蜂之间有微小差异,而二者雄蜂之间无差异,但Cytb与COI序列分析结果一致表明:E. altissima与Eupristina sp.雄蜂之间,以及二者雌蜂之间的遗传距离均差异显著,表明形态变异滞后于基因变异。雌蜂在表型上进化快于雄蜂,可能是由于雌蜂羽化后从榕果出飞,受到外界环境因素的影响较大,且两种雌蜂在传粉功能上存在差异,故二者之间的形态差异较大,而雄蜂寿命短,又终生生活在黑暗封闭、环境变化相对恒定的榕果内,两种雄蜂在行为上不存在差异,故二者表型变异较为缓慢。E. altissima和Eupristina sp.小蜂对宿主的专一性不强,在榕-蜂协同进化过程中,可能发生过宿主转移事件。     

Eimeria tenella,Eimeria necatrix, and Eimeria adenoeides: Peripheral blood leukocyte response of chickens and turkeys to strains adapted to the turkey embryo

The peripheral blood leukocyte responses of chickens and turkeys inoculated with one of three strains of a chicken Eimeria species adapted to the turkey embryo with their respective parent lines, or with E. adenoeides of the turkey were studied. The adapted lines tended to cause hematological changes in chickens and turkeys similar to those caused by E. adenoeides. These parasites caused the most significant increases in large mononuclear white blood cells = (monocytes) in both chickens and turkeys. These results provide further evidence for a monocyte/macrophage effector mechanism in the rejection of heterologous species of Eimeria from a nonspecific host. The results also agree with previous studies that show that increases in mononuclear white blood cells during parent E. tenella and E. necatrix infections in chickens occur during the periods of greatest tissue damage (3–4 days after inoculation). The generally unaffected lymphocyte numbers and increases in mononuclear white blood cells during infections with the adapted lines probably explain the reduced pathogenicity and the lack of immunogenicity seen previously in chickens inoculated with these three lines. Possibly, monocytes/macrophages play a role in the host specificity of the parasites.     

Identification of a telomeric DNA-binding protein in Eimeria tenella

Coccidiosis is considered to be a major problem for the poultry industry, and coccidiosis control is yet urgent. Due to the roles in telomere length regulation and end protection, telomere-binding proteins have been considered as a good target for drug design. In this work, a putative Gbp1p that is similar to telomeric DNA-binding protein Gbp (G-strand binding protein) of Cryptosporidium parvum, was searched in the database of Eimeria tenella. Sequence analysis indicated E.tenella Gbp1p (EtGbp1p) has significant sequence similarity to other eukaryotic Gbps in their RNA recognition motif (RRM) domains. Electrophoretic mobility shift assays (EMSAs) demonstrated recombinant EtGbp1p bound G-rich telomeric DNA, but not C-rich or double-stranded telomeric DNA sequences. Competition and antibody supershift assays confirmed the interaction of DNA–protein complex. Chromatin immunoprecipitation assays confirmed that EtGbp1p interacted with telomeric DNA in vivo. Collectively, these evidences suggest that EtGbp1p represents a G-rich single-stranded telomeric DNA-binding protein in E.tenella.     

A genetic linkage map for the apicomplexan protozoan parasite Eimeria maxima and comparison with Eimeria tenella

Eimeria maxima is one of the seven Eimeria spp. that infect the chicken and cause the disease coccidiosis. The well characterised immunogenicity and genetic diversity associated with E. maxima promote its use in genetics-led studies on avian coccidiosis. The development of a genetic map for E. maxima, presented here based upon 647 amplified fragment length polymorphism markers typed from 22 clonal hybrid lines and assembled into 13 major linkage groups, is a major new resource for work with this parasite. Comparison with genetic maps produced for other coccidial parasites indicates relatively high levels of genetic recombination. Conversion of ∼14% of the markers representing the major linkage groups to sequence characterised amplified region markers can provide a scaffold for the assembly of future genomic sequences as well as providing a foundation for more detailed genetic maps. Comparison with the Eimeria tenella genetic map produced 10 years ago has revealed a less biased marker distribution, with no more than nine markers mapped within any unresolved heritable unit. Nonetheless, preliminary bioinformatic characterisation of the three largest publicly available genomic E. maxima sequences suggest that the feature-poor/feature-rich structure which has previously been found to define the first sequenced E. tenella chromosome also defines the E. maxima genome. The significance of such a segmented genome and the apparent potential for variation in genetic recombination will be relevant to haplotype stability and the longevity of future anticoccidial strategies based upon multiple loci targeted by novel chemotherapeutic drugs or recombinant subunit vaccines.