The “self-cleaving” 2A sequence of picornavirus, which mediates ribosome-skipping events, enables the generation of two or more separate peptide products from one mRNA containing one or more “self-cleaving” 2A sequences. In this study, we introduced a single 2A sequence of porcine teschovirus-1 (P2A) linked to two fluorescent protein genes, the enhanced yellow fluorescent protein (EYFP) gene and the red fluorescent protein (RFP) gene, in a single cassette into transgenic Eimeria tenella (EtER). As expected, we obtained two separated protein molecules rather than a fused protein, although the two molecules were translated from the same mRNA carrying a single “self-cleaving” 2A sequence. Importantly, RFP led by a secretion signal was secreted into parasitophorous vacuoles, while EYFP localized mainly to the nucleus of EtER. Our results demonstrate that the “self-cleaving” 2A sequence actively mediated cleavage of polyproteins in the apicomplexan parasite E. tenella.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0351-z) contains supplementary material, which is available to authorized users.

Vaccine delivery is critical in antigen discovery and vaccine efficacy and safety. The diversity of infectious diseases in humans and livestock has required the development of varied delivery vehicles to target different pathogens. In livestock animals, previous strategies for the development of coccidiosis vaccines have encountered several hurdles, limiting the development of multiple species vaccine formulations. Here, we describe a novel vaccine delivery system using transgenic Eimeria tenella expressing immunodominant antigens of Eimeria maxima. In this delivery system, the immune mapped protein 1 of E. maxima (EmIMP1) was delivered by the closely related species of E. tenella to the host immune system during the whole endogenous life cycle. The overexpression of the exogenous antigen did not interfere with the reproduction and immunogenicity of transgenic Eimeria. After immunization with the transgenic parasite, we detected EmIMP1’s and E. maxima oocyst antigens’ specific humoral and cellular immune responses. In particular, we observed partial protection of chickens immunized with transgenic E. tenella against subsequent E. maxima infections. Our results demonstrate that the transgenic Eimeria parasite is an ideal coccidia antigen delivery vehicle and represents a new type of coccidiosis vaccines. In addition, this model could potentially be used in the development of malaria live sporozoite vaccines, in which antigens from different strains can be expressed in the vaccine strain.

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The surface antigen 1 of Toxoplasma gondii (TgSAG1) is a major immunodominant antigen and is widely considered an ideal candidate for the development of an effective recombinant vaccine against toxoplasmosis. Eimeria tenella, an affinis apicomplexan parasite with T. gondii, is a potential vaccine vector carrying exogenous antigens that stimulates specific immune responses. Here, we engineered TgSAG1 into E. tenella and obtained a stably transfected E. tenella line (Et-TgSAG1). We found TgSAG1 localized on the cell surface of Et-TgSAG1, which is similar to its native distribution in T. gondii tachyzoites. We immunized the chickens with Et-TgSAG1 orally and detected TgSAG1-specific immune responses, which partly reduced T. gondii infection. In the mouse model, we immunized the mice with Et-TgSAG1 sporozoites intraperitoneally and challenged them with T. gondii tachyzoites RH strain. We found that the mice immunized with Et-TgSAG1 showed a TgSAG1 specific Th 1-dominant immune response and a prolonged survival time compared with wild-type E. tenella and non-immunized mice. Collectively, our results demonstrated that Et-TgSAG1, utilized as a recombinant vaccine against toxoplasmosis, could be applied in both chickens and mice. Our findings also provide a promising persuasion for the development of transgenic Eimeria as vaccine vectors for use in birds and mammals.

Molecular identification of Eimeria parasites infecting poultry and livestock has been commonly used for more than 20 years. An important step of the molecular identification technique is the rupturing of the oocyst wall for DNA extraction. Previously, DNA extraction methods included pre-treatment with sodium hypochlorite and osmotic shock with saturated salt solution. Here, we present a modification of this technique for a more sensitive and efficient identification of Eimeria spp. in field samples. The disruption extent of the oocyst walls, yield of DNA extraction, and identification of species-specific DNA sequences by PCR were used to evaluate this optimized method. Incubation of oocysts in sodium hypochlorite for 1.5 h at 4 °C followed by treatment with a saturated salt solution for 1 h at 55 °C broke up the walls of most Eimeria tenella oocysts, as well as other coccidian species of chicken and rabbit, such as Eimeria intestinalis and even Cryptosporidium cuniculus. Notably, polymerase chain reaction (PCR) amplification of the intervening transcribed sequence 1 (ITS-1) was successfully performed with genomic DNA extracted from just 50 oocysts using this optimized method. Our findings will greatly promote the development of molecular diagnosis methods of coccidiosis and simplify coccidian species identification and categorization as well as infection prevalence, providing a significant advancement in the development of techniques for coccidiosis control and prevention.