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.     

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.     

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.     

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.     

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.     

The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link.We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.     

蝴蝶兰属植物杂交育种研究进展

对蝴蝶兰属(Phalaenopsis)植物的种质资源, 杂交育种过程中亲本的选择和某些观赏性状的遗传规律、杂交组合登录情况等进行了综述,并且对我国杂交育种中存在的问题进行了探讨,最后结合蝴蝶兰属植物遗传育种现状,就如何加快我国蝴蝶兰属植物杂交育种和新品种培育的进程提出了建议。     

Toxoplasma gondii: Protective effect of an intranasal SAG1 and MIC4 DNA vaccine in mice

Infections by the intracellular protozoan parasite Toxoplasma gondii are widely prevalent in humans and other animals which can cause severe or lethal toxoplasmosis. So the development of a more effective vaccine is needed urgently. A multiantigenic vaccine against toxoplasmosis was constructed in the present study, which contains two T. gondii antigens, SAG1 and MIC4 on the basis of previous immunological and immunization studies. The eukaryotic plasmid pcDNA3.1-SAG1-MIC4, pcDNA3.1-SAG1, pcDNA3.1-MIC4 were constructed first, which can express surface protein SAG1 and microneme protein MIC4 from different stages of T. gondii life cycle, and the expression ability of these DNA vaccine in HeLa cells were examined by Western blot. The efficacy of these plasmids with or without co-administration of a plasmid encoding cholera toxin A2/B as a genetic adjuvant by mucosal way to protect BALB/c mice against toxoplasmosis was evaluated. We found these vaccines were able to elicit a significant humoral and cellular immune response in vaccinated mice and they can increase survival rate and prolong the life of mice that were infected by T. gondii especially in the pcDNA3.1-SAG1-MIC4 group. Co-delivery of cholera toxin A2/B further enhanced the potency of multiantigenic DNA vaccine by intranasal route. These results encourage further research towards achieving vaccinal protection against the T. gondii in animals and humans.     

Different sensitivity of Phragmites australis and Glyceria maxima to high availability of ammonium-N

The ability to cope with NH₄⁺-N was studied in the littoral helophytes Phragmites australis and Glyceria maxima, species commonly occupying fertile habitats rich in NH₄⁺ and often used in artificial wetlands. In the present study, Glyceria growth rate was reduced by 16% at 179 μM NH₄⁺-N, and the biomass production was reduced by 47% at 3700 μM NH₄⁺-N compared to NO₃⁻-N. Similar responses were not found in Phragmites. The amounts (mg g⁻¹ dry wt) of starch and total non-structural carbohydrates (TNC) in rhizomes were significantly lower in NH₄⁺ (8.9; 12.2 starch; 20.1; 41.9 TNC) compared to NO₃⁻ treated plants (28.0; 15.6 starch; 58.5; 56.3 TNC) in Phragmites and Glyceria, respectively. In addition, Glyceria showed lower amounts (mg g⁻¹ dry wt) of soluble sugars, TNC, K⁺, and Mg²⁺ in roots under NH₄⁺ (5.6; 14.3; 20.6; 1.9) compared to NO₃⁻ nutrition (11.6; 19.9; 37.9; 2.9, for soluble sugars, TNC, K⁺, and Mg²⁺, respectively), while root internal levels of NH₄⁺ and Ca²⁺ (0.29; 4.6 mg g⁻¹ dry wt, mean of both treatments) were only slightly affected. In Phragmites, no changes in soluble sugars, TNC, Ca²⁺, K⁺, and Mg²⁺ contents of roots (7.3; 14.9; 5.1; 17.3; 2.6 mg g⁻¹ dry wt, means of both treatments) were found in response to treatments. The results, therefore, indicate a more pronounced tolerance towards high NH₄⁺ supply in Phragmites compared to Glyceria, although the former may be susceptible to starch exhaustion in NH₄⁺-N nutrition. In contrast, Glyceria's ability to colonize fertile habitats rich in NH₄⁺ is probably related to the avoidance strategy due to shallow rooting or to the previously described ability to cope with high NH₄⁺ levels when P availability is high and NO₃⁻ is also provided.     

Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala

Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 °C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.     

Transfection of Eimeria and Toxoplasma using heterologous regulatory sequences

Eimeriatenella and Toxoplasmagondii are Apicomplexan protozoa and share many similarities in biology and genomics. While the latter parasites are easily cultured in vitro and genetically manipulated, many Eimeria species are difficult to grow in vitro. We hypothesised that molecular tools for the genetic manipulation of T. gondii could be applied to the study of Eimeria parasites. Here we show that three different promoter sequences originating from E. tenella could function effectively not only in other species of the Eimeria genus (histone H4) but also in T. gondii (histone H4, actin and tubulin). Similarly, promoters of the “housekeeping” gene (tubulin) and differentially regulated gene (surface antigen gene, sag1) of T. gondii were effective in driving the expression of the yellow fluorescent protein (YFP) maker gene in E. tenella. The transfection efficiency with heterologous regulatory sequences was similar to that with homologous promoters; while the promoter strength of heterologous vectors is slightly weaker than the homologous vectors in both E. tenella and T. gondii. The results suggest that 5′ regulatory sequences are functionally conserved not only among the Eimeria species, but also between T. gondii and E. tenella, and that T. gondii could be used as a novel transfection check system for Eimeria-rooted vectors, accelerating the development of reverse genetics in Eimeria spp.     

Eimeria acervulina,E. brunetti,E. maxima, and E. necatrix: Low doses of oocysts to immunize young chickens

Resistance to reinfection varied with the species of Eimeria and with the number of oocysts in the inoculum. Chickens immunized with doses of 20,000 and 80,000 oocysts of E. acervulina, 312 and 1250 oocysts of E. brunetti or E. necatrix, or 1250 and 5000 oocysts of E. maxima at 2 and 4 weeks of age, respectively, were almost completely immune to a challenge dose at 6 weeks of age. Resistance was slightly less in chickens immunized with 1250 and 5000 oocysts of E. acervulina or 312 and 1250 oocysts of E. maxima. Birds given three immunizing infections of 1250, 5000, and 20,000 oocysts of E. maxima were completely immune 8 weeks after the last dose. Resistance was slightly less in birds immunized with similar doses of E. brunetti or E. necatrix. Doses of 20,000, 80,000, and 320,000 oocysts appeared necessary to confer a high level of immunity to E. acervulina. More than three low doses of oocysts appear necessary to induce a complete and enduring immunity against a high challenge for E. acervulina, E. brunetti, and E. necatrix. Higher immunizing doses would not be satisfactory due to the pathogenic effects of the coccidia after the initial infection.     

Haplorchis pumilio and H. taichui in Vietnam discriminated using ITS-2 DNA sequence data from adults and larvae

Samples of Haplorchis taichui and Haplorchis pumilio of different life-stages (cercariae, metacercariae and adults) and from different host species (snail, fish, dog, cat and human) were collected in Nghe An and Nam Dinh Provinces in Vietnam. Samples from Thailand were available for comparison. All adults and metacercariae were initially identified using morphological criteria. Polymerase chain reaction (PCR) assays were developed for discriminating between the species. The complete sequence for the nuclear ribosomal internal transcribed spacer-2 (ITS-2) was obtained from one adult and one metacercaria of H. taichui and three adults and three metacercariae of H. pumilio from Vietnam. Sequences from cercariae from three different snails clustered with those of H. pumilio. Intra-individual variation in the ITS-2 region was detected by sequencing of cloned PCR products. These are the first sequences from Vietnamese Haplorchis spp. to be reported and demonstrate that H. taichui and H. pumilio can be identified unambiguously from any life-stage, including the cercarial stage that is difficult to identify using morphology. Discrepancies in the literature are discussed and examples of apparent misidentification highlighted. The data provide a resource to assist in taxonomic studies on heterophyids, in the design of probes for diagnosis and for field surveys to identify infection in snails.     

Eimeria tenella: Effects of diclazuril treatment on microneme genes expression in second-generation merozoites and pathological changes of caeca in parasitized chickens

The effects of diclazuril on mRNA expression levels of invasion-related microneme genes were examined in second-generation merozoites of Eimeria tenella (E. tenella) by quantitative real-time (QRT) PCR. Diclazruil treatment of infected chickens significantly decreased the number of second-generation merozoites by 65.13%, and resulted in downregulation of EtMIC genes: EtMIC1 by 65.63%, EtMIC2 by 64.12%, EtMIC3 by 56.82%, EtMIC4 by 73.48%, and EtMIC5 by 78.17%. SEM images of caecum tissue from uninfected chickens showed regular intestinal villus structure. In infected chickens, a distinct loss of the superficial epithelium, with a flattened mucosa and large-area necrosis and anabrosis, was evident. In diclazruil-treated chickens, a decrease in merozoite number and a visibly improved appearance of the caeca were noted. These improvements appeared to be mediated in part by downregulation of the expression of invasion-related EtMIC genes in response to diclazuril.