A rhoptry protein, localizing in the bulb region of rhoptries, could induce protective immunity against Eimeria tenella infection.

Background: Rhoptry organelle proteins (ROPs) secreted by apicomplexan parasites play important roles during parasites invasion and survival in host cells, and are potential vaccine candidates against apicomplexan diseases. Eimeria tenella (E. tenella) is one of the most noteworthy apicomplexan species, which causes hemorrhagic pathologies. Although dozens of putative E. tenella ROP sequences are annotated, most ROP proteins are not well studied. Methods: In this study, an E. tenella ROP21 gene was identified and the recombinant EtROP21 protein (rEtROP21) was expressed in Escherichia coli. The developmental expression levels, localization, and protective efficacy against E. tenella infection in chickens were studied. Results: An EtROP21 gene fragment with an open reading frame (ORF) of 981 bp was obtained from the Beijing strain of E. tenella. The rEtROP21 has a molecular weight of approximately 50 kDa and was recognized by rEtROP21-immunized mouse serum. Two specific protein bands, about 43 KDa and 95 KDa in size, were detected in the whole sporozoite proteins using the rEtROP21-immunized chicken serum. RT-qPCR analysis of the E. tenella ROP21 gene (EtROP21) revealed that its mRNA levels were higher in merozoites and sporozoites than in sporulated and unsporulated oocysts. Immunofluorescence and immunoelectron analyses showed that the EtROP21 protein predominantly localizes in the bulb region of rhoptries distributed at anterior, posterior, and perinuclear regions of E. tenella sporozoites. Immunization and challenge experiments revealed that immunizing chickens with rEtROP21 significantly increased their average body weight gain while decreasing mean lesion score and oocyst output (P <0.05). When compared with the challenged control group, the rEtROP21-immunized group was associated with a significantly higher relative weight gain (90.2%) and a greater reduction in oocyst output (67%) (P <0.05). The anticoccidial index of the rEtROP21-immunized group was 163.2. Chicken serum ELISA revealed that the levels of the specific anti- rEtROP21 antibody, IFN-γ, and IL-4 were significantly higher in the rEtROP21-immunized group than in the challenged control group (P <0.05). Conclusion: These results indicate that rEtROP21 can induce a high level of specific immune response and it is a potential candidate for the development of vaccines against E. tenella infection in chickens.

Molecular characterization and protective efficacy of vacuolar protein sorting 29 from Eimeria tenella.

Introduction: Vacuolar protein sorting 29 (VPS29) is a core component of the retromer-retriever complex and is essential for recycling numerous cell-surface cargoes from endosomes. However, there are no reports yet on VPS29 of Eimeria spp. Methods: Here, we cloned and prokaryotically expressed a partial sequence of Eimeria tenella VPS29 (EtVPS29) with RT-PCR and engineered strain of Escherichia coli respectively. The localization of the VPS29 protein in E. tenella sporozoites was investigated with immunofluorescence (IFA) and overexpression assays. And its protective efficacy against E. tenella infection was investigated in chickens with the animal protection test. Results: An EtVPS29 gene fragment with an ORF reading frame of 549 bp was cloned. The band size of the expressed recombinant protein, rEtVPS29, was approximately 39 kDa and was recognized by the chicken anti-E. tenella positive serum. EtVPS29 protein was observed widely distributing in the cytoplasm of E. tenella sporozoites in the IFA and overexpression assays. rEtVPS29 significantly increased average body weight gain and decreased mean lesion score and oocyst output in chickens. The relative weight gain rate in the rEtVPS29-immunized group was 62.9%, which was significantly higher than that in the unimmunized and challenged group (P < 0.05). The percentage of reduced oocyst output in the rEtVPS29 immunized group was 32.2%. The anticoccidial index of the rEtVPS29-immunized group was 144.2. Serum ELISA also showed that rEtVPS29 immunization induced high levels of specific antibodies in chickens. Discussion: These results suggest that rEtVPS29 can induce a specific immune response and is a potential candidate for the development of novel vaccines against E. tenella infections in chickens.

Protective efficacy of Toxoplasma gondii GRA12 or GRA7 recombinant proteins encapsulated in PLGA nanoparticles against acute Toxoplasma gondii infection in mice.

Background: Toxoplasma gondii is an apicomplexan parasite that affects the health of humans and livestock, and an effective vaccine is urgently required. Nanoparticles can modulate and improve cellular and humoral immune responses. Methods: In the current study, poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles were used as a delivery system for the T. gondii dense granule antigens GRA12 and GRA7. BALB/c mice were injected with the vaccines and protective efficacy was evaluated. Results: Mice immunized with PLGA+GRA12 exhibited significantly higher IgG, and a noticeable predominance of IgG2a over IgG1 was also observed. There was a 1.5-fold higher level of lymphocyte proliferation in PLGA+GRA12-injected mice compared to Alum+GRA12-immunized mice. Higher levels of IFN-g and IL-10 and a lower level of IL-4 were detected, indicating that Th1 and Th2 immune responses were induced but the predominant response was Th1. There were no significant differences between Alum+GRA7-immunized and PLGA+GRA7-immunized groups. Immunization with these four vaccines resulted in significantly reduced parasite loads, but they were lowest in PLGA+GRA12-immunized mice. The survival times of mice immunized with PLGA+GRA12 were also significantly longer than those of mice in the other vaccinated groups. Conclusion: The current study indicated that T. gondii GRA12 recombinant protein encapsulated in PLGA nanoparticles is a promising vaccine against acute toxoplasmosis, but PLGA is almost useless for enhancing the immune response induced by T. gondii GRA7 recombinant protein.

Pathogenicity and drug resistance of the Eimeria tenella isolate from Yiwu, Zhejiang province, eastern China.

Chicken coccidiosis can cause severe enteritis with high mortality, which causes serious economic losses to the global breeding industry each year. The most virulent species is Eimeria tenella (E. tenella), but the infectivity of different E. tenella varies among geographic strains. At present, there are no reports related to the pathogenicity and drug resistance of E. tenella in Yiwu, Zhejiang province, China. A total of 600 fecal samples were collected from 10 farms in Zhejiang province, the overall oocyst prevalence was 54.2% (325/600). The prevalence was significantly higher (P < 0.01) in chickens under 40 d (97.5%) than that in chickens between 60 and 85-days-old (40.5%) and chickens over 90-days-old (24.5%). E. tenella stain was isolated from fecal samples of chickens in Yiwu and the pathogenicity of this isolate was determined, and then we recorded the survival rate, bloody stool score, lesion score, average weight gain. The results showed that all of the chickens infected with 5 × 105 sporulated oocysts of E. tenella died after the seventh day of infection, the bloody stool score and average lesion score of chickens from group 1 (5 × 105), group 2 (5 × 104), group 3 (5 × 103) and group 4 (5 × 102) decreased successively; the average weight gain (g) and relative weight gain (%) increased successively; the weight gain of the low-dose E. tenella infection groups (5 × 103 and 5 × 102) were higher than the other 2 groups (5 × 105 and 5 × 104) (P < 0.05). Finally, The E. tenella isolate was tested for sensitivity to 6 anticoccidial drugs (sulfachloropyrazine sodium, amproline, toltrazuril, clopidol, salinomycin, and nicarbazine) using 4 indexes including anticoccidial index(ACI), percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS), and relative oocyst production (ROP). The results showed that this isolate has developed severe resistance to drugs of salinomycin and nicarbazine, moderate resistance to amproline and clopidol, slight resistance to toltrazuril, while the E. tenella isolate performed more sensitive to sulfachloropyrazine sodium.

Dominant infection of Cryptosporidium baileyi in broiler chickens in Zhejiang Province, China.

Cryptosporidium is a common enteric parasite in chickens. A total of 812 fecal specimens were collected from 11 broiler farms in Zhejiang Province, China, and analyzed by nested PCR amplification based on the small subunit ribosomal RNA (SSU rRNA) gene. The overall infection rate of Cryptosporidium was 6.3% (51/812), and five of 11 farms were Cryptosporidium positive. Broilers aged > 90 days accounted for the highest infection rate of 16.1% (6/56), followed by those aged 30-60 days (10.6%, 38/358) and 60-90 days (4/378, 1.1%). Two Cryptosporidium species were identified by sequence analysis, with the predominant species being C. baileyi (96.1%, 49/51) and the minor infection being C. meleagridis (3.9%, 2/51). Based on the 60-kDa glycoprotein (gp60) gene, two C. meleagridis-positive isolates were identified as one known subtype, IIIbA24G1R1. This study indicated the common occurrence of C. baileyi in broiler chickens in this region and low zoonotic transmission potential of Cryptosporidium to humans.

Molecular Detection and Genotyping of Toxoplasma gondii from Pigs for Human Consumption in Zhejiang and Jiangsu Provinces, Eastern China.

Toxoplasma gondii infections are common in humans and animals worldwide. Ingestion of raw or undercooked meat containing tissue cysts of T. gondii is one major source of transmission of this parasite. It is important to guarantee the meat quality of China since our pork industry produces about half of the world's pork. In this study, a total of 746 pig samples were collected from Zhejiang and Jiangsu provinces in eastern China, and examined for T. gondii infection by PCR amplification targeting B1 gene. In this study, we found that 57 of 746 (7.6%) pigs were positive for B1 gene, with 8.5% (48/562) in Zhejiang province and 4.9% (9/184) in Jiangsu province, respectively. The positive DNA samples were further genotyped at 11 genetic markers, including SAG1, 5'-and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico through PCR-restriction fragment length polymorphism (PCR-RFLP) technology. Two genotypes (ToxoDB 9 and ToxoDB 10) of T. gondii were identified by PCR-RFLP in Zhejiang province. However, both genotypes were not determined from Jiangsu province, which is speculated on the low DNA concentration and the small number of samples. These results indicate that T. gondii infection is endemic in pigs in eastern China and may raise public food safety concerns, suggesting more interventions for T. gondii-related risks are needed in the future.

An Investigation of the Relationship between Cyniclomyces guttulatus and Rabbit Diarrhoea.

Cyniclomyces guttulatus is usually recognised as an inhabitant of the gastrointestinal (GI) tract in rabbits. However, large numbers of C. guttulatus are often detected in the faeces of diarrhoeic rabbits. The relationship of C. guttulatus with rabbit diarrhoea needs to be clearly identified. In this study, a C. guttulatus Zhejiang strain was isolated from a New Zealand White rabbit with severe diarrhoea and then inoculated into SPF New Zealand white rabbits alone or co-inoculated with Eimeriaintestinalis, another kind of pathogen in rabbits. Our results showed that the optimal culture medium pH and temperature for this yeast were pH 4.5 and 40-42 °C, respectively. The sequence lengths of the 18S and 26S ribosomal DNA fragments were 1559 bp and 632 bp, respectively, and showed 99.8% homology with the 18S ribosomal sequence of the NRRL Y-17561 isolate from dogs and 100% homology with the 26S ribosomal sequence of DPA-CGR1 and CGDPA-GP1 isolates from rabbits and guinea pigs, respectively. In animal experiments, the C. guttulatus Zhejiang strain was not pathogenic to healthy rabbits, even when 1 × 108 vegetative cells were used per rabbit. Surprisingly, rabbits inoculated with yeast showed a slightly better body weight gain and higher food intake. However, SPF rabbits co-inoculated with C. guttulatus and E. intestinalis developed more severe coccidiosis than rabbits inoculated with C. guttulatus or E. intestinalis alone. In addition, we surveyed the prevalence of C. guttulatus in rabbits and found that the positive rate was 83% in Zhejiang Province. In summary, the results indicated that C. guttulatus alone is not pathogenic to healthy rabbits, although might be an opportunistic pathogen when the digestive tract is damaged by other pathogens, such as coccidia.

Enhancing Immune Responses to a DNA Vaccine Encoding Toxoplasma gondii GRA7 Using Calcium Phosphate Nanoparticles as an Adjuvant.

Toxoplasma gondii infects almost all warm-blooded animals, including humans. DNA vaccines are an effective strategy against T. gondii infection, but these vaccines have often been poorly immunogenic due to the poor distribution of plasmids or degradation by lysosomes. It is necessary to evaluate the antigen delivery system for optimal vaccination strategy. Nanoparticles (NPs) have been shown to modulate and enhance the cellular humoral immune response. Here, we studied the immunological properties of calcium phosphate nanoparticles (CaPNs) as nanoadjuvants to enhance the protective effect of T. gondii dense granule protein (GRA7). BALB/c mice were injected three times and then challenged with T. gondii RH strain tachyzoites. Mice vaccinated with GRA7-pEGFP-C2+nano-adjuvant (CaPNs) showed a strong cellular immune response, as monitored by elevated levels of anti-T. gondii-specific immunoglobulin G (IgG), a higher IgG2a-to-IgG1 ratio, elevated interleukin (IL)-12 and interferon (IFN)-γ production, and low IL-4 levels. We found that a significantly higher level of splenocyte proliferation was induced by GRA7-pEGFP-C2+nano-adjuvant (CaPNs) immunization, and a significantly prolonged survival time and decreased parasite burden were observed in vaccine-immunized mice. These data indicated that CaPN-based immunization with T. gondii GRA7 is a promising approach to improve vaccination.

Transcriptome Analysis Reveals the Molecular Mechanism of Resting Cyst Formation in Colpoda aspera.

Resting cyst formation is a remarkable survival strategy used by ciliates in response to the adverse environmental conditions. However, the mechanisms underlying encystment are poorly understood. Here, the genetic basis of encystment in Colpoda aspera was examined through RNA sequencing to identify transcriptome-wide changes in gene expression between vegetative and encystment stages. After de novo assembly, 49,543 transcripts were identified. Gene annotation and pathway mapping analysis revealed marked changes in biosynthesis, energy metabolism, and autophagy pathways during cyst formation. In addition, some differentially regulated genes were predicted to function in the interconnected cAMP, AMPK, mTOR, and PI3K/AKT signaling pathways, potentially forming a regulatory network for encystment. The present study conducted a large-scale assessment of Colpoda aspera genomic resources and provides new insight into the molecular mechanisms underlying cyst formation.

Identification of a novel Hemoplasma species from pigs in Zhejiang province, China.

Hemoplasmas belong to Mycoplasmataceae (Mollicutes: Mycoplasmatales) and are able to infect a broad range of mammalian species. We investigated prevalence of hemotropic mycoplasma species in pig farms in the region of Zhejiang by a PCR scheme using universal primers targeting 16S rRNA and RNase P RNA gene (rnpB). Representative positive samples from different farms were selected for sequencing of 16S rRNA and the 219bp rnpB gene fragments for phylogenetic analysis. Sequencing analysis of PCR products from first samples identified a novel hemoplasma species present in several pig farms in the region with highest nucleotide identity of 92% to Candidatus Mycoplasma turicensis. A duplex PCR assay was then designed for differential detection of the novel hemoplasma from Mycoplasma parvum/M. suis in field samples. Of 324 blood samples from clinically healthy pigs, 26.5% was positive for this novel hemoplasma species and 50% positive for M. suis/M. parvum, indicating that the novel hemotropic mycoplasma species were of considerably high prevalence in Zhejiang province, China.

Transgenic Eimeria magna Pérard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.).

Rabbit coccidiosis causes great economic losses to world rabbitries. Little work has been done considering genetic manipulation on the etiological agents, rabbit Eimeria spp. In this study, we constructed a transgenic line of Eimeria magna (EmagER) expressing enhanced yellow fluorescent protein (EYFP) and red fluorescent protein (RFP) using regulatory sequences of Eimeria tenella and Toxoplasma gondii. We observed the life cycle of EmagER and confirmed that the transgenic parasites express exogenous proteins targeted to different cellular compartments throughout the entire life cycle. EYFP was expressed mainly in the nucleus and RFP both in the nucleus and cytoplasm. Then, coccidia-free, laboratory-reared 40-day-old rabbits were primarily infected with either EmagER or wild-type strain oocysts and challenged with the wild-type strain. EmagER showed similar reproductivity and immunogenicity to the wild-type strain. Finally, we examined the foreign protein-specific immune response elicited by EmagER. Rabbits were immunized with either transgenic or wild-type oocysts. Immune response against parasite-soluble antigen, EYFP and RFP in spleen, and mesenteric lymph nodes were detected by quantitative real-time PCR. The relative expression level of IFN-γ, IL-2, and TNF-α were higher in EmagER-immunized rabbits than wild-type parasites-immunized rabbits after stimulation with EYFP and RFP. Our study confirmed that a specific immune response was induced by the exogenous protein expressed by EmagER and favored future studies on application of transgenic rabbit coccidia as recombinant vaccine vectors.

Stable Transfection of Eimeria intestinalis and Investigation of Its Life Cycle, Reproduction and Immunogenicity.

Rabbit coccidiosis, caused by infection of Eimeria spp. is one of the most severe parasitic diseases in rabbits. Eimeria intestinalis is one of the most immunogenic species in rabbit coccidia. Due to the lack of genomic information and unsuccessful in vitro cultivation, genetic manipulation of rabbit coccidia lagged behind other apicomplexan parasites. Using regulatory sequences from E. tenella, we obtained a transgenic line of E. intestinalis expressing yellow fluorescent protein (YFP). YFP was continuously expressed throughout the whole life cycle. Morphological features of E. intestinalis in different developmental stages were dynamically observed with the transgenic line. Some important features in the endogenous development stages were observed. Trophozoites were found as early as 4 h post inoculation. Two types of schizonts and merozoites were observed in first three of the four schizogonies. Beside jejunum and ileum, gametogony stage and oocysts were also found in the duodenum and vermiform appendix. In addition, the transgenic strain was highly immunogenic but less pathogenic than the wild type. Considering the high immunogenicity of E. intestinalis and amenability to transfection with foreign genes, transgenic E. intestinalis could be a promising oral eukaryotic vaccine vector.

Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters.

The promoter plays an important role in the regulation of gene expression. To analyze a promoter's activity, we developed a novel lentiviral T/A vector that contains two reporter genes, a luciferase (Luc2) gene and a green fluorescent protein (Venus) gene, that are linked via an internal ribosome entry site (IRES2). To test the performance of this vector, phosphoglycerate kinase-1 (PGK) and elongation factor-1α (EF1α) promoters were amplified by PCR and inserted into this lentiviral T/A vector using T4 DNA ligase, yielding two promoter-reporter vectors: pLent-T-PGK and pLent-T-EF1α. When these vectors were transfected into 293T cells, we observed a higher level of Venus expression under a fluorescence microscopy in the case of pLent-T-EF1α as compared to pLent-T-PGK. The results of the luciferase reporter assay showed that the ratio of the promoter activities of EF1α and PGK was approximately 9:1. The two promoter-reporter vectors were also packaged as lentiviral particles to conduct promoter activity assay in cultured cells. The ratio of the promoter activities of EF1α and PGK was 4.23:1 when they were infected into 293T cells at a multiplicity of infection of 1. This value is comparable to that of a parallel experiment using the commercial luciferase reporter vector pGL4.10 with an activity ratio of 5.99:1 for EF1α and PGK. These results indicate that lentiviral T/A vector will be a useful tool for analysis of promoter activity and specificity.

[Preparation and immunogenicity of silk fibroin/chitosan microspheres for DNA vaccine delivery against infectious bursal disease virus].

To evaluate the immunities of biodegradable microsphere as a release delivery system for DNA vaccine against Infectious Bursal Disease Virus, in our study, silk fibroin/chitosan microsphere adjuvant was prepared with a precipitation/coacervation method. Both glutaraldehyde and Na2SO4 solution were used in cross-linking. No immune chicken were intramuscularly inoculated at 14 day-old and boosted 2 weeks later. The results show that glutaraldehyde destroyed the DNA activity of the vaccine whereas Na2SO4 solution did not. Factors of the chitosan concentration 0.5% (pH 5.0), silk fibroin concentration 0.6%, plasmid DNA (500 microg/mL) dissolved in 2% Na2SO4 solution were optimized to produce microsphere, with a loading capacity of 89.14%. The average particle size of SF-CS/pCI-VP2/4/3 microsphere is 1.98 microm, and it can protect the loading DNA vaccine from DNase I digestion. Data from anti IBDV ELISA antibodies in the serum show that immunization activity of the microsphere groups were generally higher than plasmid vaccine group (P < 0.05), and the SF/CS compound microspheres group was better than that of sole CS microsphere group. The developed SF/CS microspheres are a very promising vaccine delivery system.

A low-virulence Eimeria intestinalis isolate from rabbit (Oryctolagus cuniculus) in China: molecular identification, pathogenicity, and immunogenicity.

An Eimeria intestinalis isolated from a rabbit in China was first identified by amplifying the 18S small subunit (SSU) ribosomal RNA gene. The size of the amplified fragment was 1521 bp. The 18S SSU RNA gene of the E. intestinalis isolate shared 99% sequence identity with E. intestinalis isolates from France and the Czech Republic, with 100 and 96% coverage, respectively. Then, the pathogenicity and immunogenicity of the E. intestinalis isolate were evaluated in specific pathogen free (SPF) rabbits. In the pathogenicity assay, SPF rabbits in four groups were infected with 5 × 10(3), 5 × 10(4), 5 × 10(5), and 0 sporulated oocysts, respectively. Clinical signs including diarrhoea, constipation, loss of appetite, and reduction of body weight gain were observed in rabbits inoculated with 5 × 10(4) and 5 × 10(5) oocysts. And one rabbit (25 %) inoculated with 5 × 10(5) oocysts died 15 days after the inoculation. In the immunogenicity assay, SPF rabbits in five groups (named B1, B2, B3, B4, and B5) were immunised with 5 × 10(1), 5 × 10(2), 5 × 10(3), 0, and 0 sporulated oocysts, respectively. All rabbits but the B5 group were challenged with 1 × 10(6) oocysts. After the challenge, no or slight clinical signs were seen in rabbits of the B2 and B3 groups. Compared with the control, a 69.6 and 84.5% reduction of oocyst output was observed in the B2 and B3 groups, respectively. The body weight gain of the two groups was obviously higher than that of the challenge control group. All the results show that the E. intestinalis isolate has low virulence but immunogenicity in rabbit.

A Colpoda aspera isolate from animal faeces: In vitro cultivation and identification.

A colpodean ciliate was found in the faeces of experimental rabbits. It was initially cultivated in medium mixed with 2% (w/v) rabbit faeces. Subsequently, two chemically defined media, designated CA-1 and CA-2, were found to be suitable for axenical cultivation of the ciliate. The maximum abundance of the ciliate isolate in the CA media was 1-2 × 10(5) cells/ml. The ciliate isolate was further identified with silver impregnation and molecular analysis. Features of the left oral polykinetid, somatic dikinetids, and sliverline pattern were similar to those of Colpoda aspera as described by Foissner (1993). The 18S small subunit ribosomal RNA gene of the ciliate isolate shared 99% sequence identity with that of C. aspera, with 100% coverage, and formed a sister clade in the phylogenetic tree with the reference C. aspera isolate. In addition, the trophozoite of C. aspera could proliferate over a temperature range from 25-37°C. When resting cysts were cultivated in CA-1 medium at 30-35°C, 98.2% of the trophozoites were detached from the cyst wall after 7 h.

piggyBac transposon-mediated transgenesis in the apicomplexan parasite Eimeria tenella.

piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5' and 3' ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac.

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.

Dynamic development of parasitophorous vacuole of Eimeria tenella transfected with the yellow fluorescent protein gene fused to different signal sequences from apicomplexan parasites.

Intracellular stages of Eimeria tenella reside within a membrane-bound parasitophorous vacuole (PV). PVs of apicomplexan parasites like E. tenella play important roles in nutrient acquisition, multiplication, and evasion of host immune responses. Different signal sequences from apicomplexan parasites were investigated in the transfected E. tenella for their functions in targeting yellow fluorescent protein (YFP) to subcompartments and the dynamic development of the PV of E. tenella was studied. Two 5' terminal signal sequences derived from Toxoplasma gondii GRA8 protein and Plasmodium falciparum repetitive interspersed family protein, respectively, were confirmed to target YFP to the PVs of the transfected E. tenella, suggesting that signal sequences are functionally conserved among Apicomplexa. Three structurally different types of PVs were observed during the endogenous development of the transfected E. tenella in vitro. In addition, three subcompartments in the PV, namely, membranous extensions into the host cell cytosol, membranous extensions into the vacuolar lumen, and particle-like bodies, were detected during schizogony of the parasite.

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 12h 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.