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.     

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.     

Host race formation in the meadowsweet and strawberry feeding leaf beetle Galerucella tenella

We investigated host race formation in Galerucella tenella, a leaf beetle which feeds preferentially on meadowsweet (Rosaceae) in its natural habitats, but has become an important pest on strawberry (Rosaceae) in agricultural areas. Beetles from two isolated sites (Skeppsvik in Sweden and Solf in Finland) were compared with respect to preference and performance. At Skeppsvik the beetles were found feeding on meadowsweet, while at Solf large populations only develop in strawberry plantations, despite the presence of meadowsweet.In reciprocal field transplantations and laboratory bioassays, beetles from both sites discriminated against their foreign host, using their natal plant to a significantly higher degree for both egg laying and feeding, but with one interesting exception, namely that beetles from Solf increased their use of and even preferred meadowsweet for feeding in the laboratory. This increased use of meadowsweet by adult Solf beetles in the laboratory (without conspecific competitors) may be attributed to a density-dependent strategy, whereby mothers avoid pre-emptying the nutritional resources of the plants that will host their offspring in cases where no competitors are present.Larval fitness did not differ significantly between host plants, although larval survival of Solf beetles was halved when reared on their non-natal host plant (meadowsweet). Although beetles from both sites preferred to oviposit on their local host plant, our results provide little evidence that this presumed adaptation has to do with the nutritional quality of the plants involved. Instead, we suggest that other factors associated with the plants, such as enemy-free space and resistance to drought may be more important selective agents, shaping host preference in the field.     

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.     

Observations on the endogenous stages of Eimeria crandallis in domestic lambs (Ovis aries)

Lambs reared coccidia-free were inoculated orally with various numbers of sporulated oocysts of E. crandallis and were killed between 1 and 22 days after inoculation; tissues were examined histologically. Sporozoites were seen 1, 2 and 3 days after inoculation (DAI) in crypt epithelial cells in the mid-jejunum. Infected cells migrated into the lamina propria where the parasite within them developed into a firstgeneration meront containing about 250,000 merozoites at 10 DAI. A second generation of meronts was seen at 10–12 DAI, each containing up to about 10 merozoites, situated mainly at the bases of crypts in the jejunum and ileum but also in the caecum. From 11 DAI pro-gamonts were seen which were enveloped by the host cell nucleus and which divided in synchrony with the host cell for an undetermined number of generations. Mature gamonts began to develop from them by 16 DAI. Oocyst output began at 16 DAI and rose to a peak at about 22 DAI. Up to 10⁸ oocysts were produced per oocyst inoculated. They showed wide variation in size and colour.     

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.     

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.     

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.     

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。     

Tomato SNP Discovery by EST Mining and Resequencing

Many economically important crop species are relatively depauparate in genetic diversity (e.g., soybean, peanut, tomato). DNA polymorphism within cultivated tomato has been estimated to be low based on molecular markers. Through mining of more than 148,000 public tomato expressed sequence tags (ESTs) and full-length cDNAs, we identified 764 EST clusters with potential single nucleotide polymorphisms (SNPs) among more than 15 tomato lines. By sequencing regions from 53 of these clusters in two to three lines, we discovered a wealth of nucleotide polymorphism (62 SNPs and 12 indels in 21 Unigenes), resulting in a verification rate of 27.2% (28 of 103 SNPs predicted in EST clusters were verified). We hypothesize that five regions with 1.6–13-fold more diversity relative to other tested regions are associated with introgressions from wild relatives. Identifying polymorphic, expressed genes in the tomato genome will be useful for both tomato improvement and germplasm conservation.     

Effects of experimental infection with Eimeria bovis on the balance of sodium, potassium and water in calves

Balance trials were performed to investigate the effects of experimental Eimeria bovis coccidiosis on the metabolism of water, sodium and potassium in calves. Non-infected pair-fed controls and controls fed according to plan were included in the study to allow differentiation between the effects due to infection and due to changes in feed intake. Primary infection with 5 × 10⁴ (group A) or 1 × 10⁵ (group B) oocysts caused mild diarrhoea in three out of four group A calves and mild to severe haemorrhagic diarrhoea in all five group B calves. Losses of sodium and potassium via faeces tended to increase in the infected calves during patency and apparent digestibility (AD) of these minerals was comparably low. In the urine of the infected calves the Na/K-ratio decreased due to a reduced urinary excretion of sodium. The retention (RT) of sodium was particularly high in the calves that had received the higher oocyst dose. Potassium RT did not underlie significant changes during the course of coccidiosis. In the infected calves the plasma level of sodium was reduced transiently while the level of potassium remained fairly stable. Infections with the higher oocyst dose caused a distinct reduction of fluid excretion via urine which compensated for the increased faecal water losses during severe diarrhoea. Reinfection of the group A calves with 1 × 10⁵ oocysts did not cause any significant metabolic impairment. The results of this study indicate that although acute sublethal bovine coccidiosis alters electrolyte and water metabolism the overall balance of electrolytes and water is largely maintained by physiologic adaptation.     

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.     

基于联合测序分析技术挖掘红苞凤梨lncRNA信息

为了揭示lncRNA在红苞凤梨嵌合叶片形成和生长发育过程中的调控作用机制,该文以金边红苞凤梨为材料,采用Hiseq2500测序和SMRT三代全长转录组测序联合测序分析技术,分析挖掘红苞凤梨lncRNA信息.结果表明:(1)鉴定得到6018条lncRNA,包含3298个基因间lncRNA,870个反义lncRNA,717...     

基于转录组测序的姬松茸镉胁迫下内参基因筛选

[目的] 筛选不同实验条件下,尤其是镉胁迫下的姬松茸内参基因,为研究姬松茸镉富集相关基因功能研究奠定基础。[方法] 根据不同浓度Cd（0、2、5 mg/L）胁迫下菌丝中转录组表达量数据,筛选出18个候选基因,设计了30对引物;根据引物扩增的特异性、扩增效率初步筛选出来自不同基因的5对引物;运用qRT-PCR检测了5个基因在不同浓度Cd胁迫下的菌丝样品、原基、菌柄和菌盖中的C_t值,利用geNorm、NormFinder和BestKeeper对这些基因的表达稳定性进行评价。[结果] SGT2和STK是菌丝阶段Cd胁迫下最稳定的2个候选内参基因,GAPDH是不同组织中最稳定的内参基因,SGT2、STK和GAPDH是所有实验条件下最稳定的3个内参基因。[结论] 在姬松茸镉胁迫条件下,本研究筛选出的SGT2和STK比常用的内参基因表现出更强的稳定性。     

Development and interspecific transferability of genic microsatellite markers in Spartina spp. with different genome size

Flow cytometry analysis showed variation of nuclear DNA content among different species of Spartina. Spartina alterniflora had the biggest genome (1763.9 Mbp) and S. cynosuroides had the smallest genome (756.35 Mbp), whereas the genomes of S. patens (969.36 Mbp) and S. spartinae (979.78 Mbp) were comparable. Mining simple sequence repeats (SSR) from 1227 expressed sequence tags (EST) generated from salt stressed S. alterniflora showed an abundance of di- and tri-nucleotide repeats. Of 100 ESSR (EST-derived SSR) loci with five or more repeats, 81 loci were successfully amplified in eight S. alterniflora genotypes and 15 (22.2%) ESSR markers were polymorphic. Eleven of the 15 polymorphic ESSRs showed amplification across six different species of Spartina while 100% cross transferability was observed with at least one species of Spartina. The average number of alleles per marker was 3.9 and 5.8 within S. alterniflora and among Spartina species, respectively. The ESSR markers discriminated different members within and between species of Spartina genus.