Tuanyuan Shi

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.

Restriction enzyme-mediated transfection improved transfection efficiency in vitro in Apicomplexan parasite Eimeria tenella

Genetic manipulation of Apicomplexan parasite Eimeria tenella is only in its earliest stages. In the current study, transfection of E. tenella was conducted by electroporating sporozoites along with linear or circular plasmid DNA, and with or without restriction enzyme. Transfection system containing both linear DNA and restriction enzyme resulted in a transfection efficiency of 2.2x10(-3)in vitro, which is 200-fold higher than that using circular plasmid DNA alone. In another transfection strategy, PCR amplicons of expression cassette, instead of whole plasmid DNA, were subjected to transfection, and it was also found successful. These results suggest that linear DNA and restriction enzyme together in the transfection system greatly improve the transfection efficiency of E. tenella. The high transfection efficiency makes possible the establishment of stable transfection in vivo; and the success of PCR-based, restriction enzyme-mediated transfection will further simplify the transfection process for E. tenella and other Apicomplexan parasites.

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.

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.

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.