The potential of Eimeria parasites as live vaccine vectors has been reported with successful genetic manipulation on several species like E. tenella, E. mitis and E. necatrix. Among seven Eimeria species infecting chickens, E. acervulina is a highly prevalent, moderately pathogenic species. Thus, it is valuable for the study of transfection and for use as a potential as vaccine vector. In this study, a plasmid containing expression cassette with enhanced yellow fluorescent protein (EYFP), red fluorescent protein (RFP) and 12 copies of extracellular domain of H9N2 avian influenza virus M2 (M2e) protein was used for the transfection. Nucleofected sporozoites were inoculated into birds through wing vein. Recombinant E. acervulina oocysts with 0.1% EYFP+ and RFP+ populations were collected from the feces of the inoculated birds. The fluorescent rate of transgenic parasites reached over 95% after nine successive propagations with a pyrimethamine selection in vivo and fluorescent-activated cell sorting (FACS) of progeny oocysts. The expression of M2e in the transgenic parasites (EaM2e) was confirmed by Western blot and its cytoplasm localization in sporozoites was displayed by an indirect immunofluorescent assay (IFA). Meanwhile, we found that the fecundity of EaM2e was equivalent to that of wild type E. acervulina (EaWT). Taken together, the stable transfection of E. acervulina was successfully established. Future studies will focus on whether transgenic E. acervulina can serve as a live vaccine vector.
Eimeria, a cousin of malarial parasites, causes coccidiosis that results in huge losses in the poultry industry. Although live coccidiosis vaccines have been developed and used widely for the successful control of the disease, the mechanism underlying protective immunity remains largely unknown. Using Eimeria falciformis as a model parasite, we observed that tissue-resident memory CD8+ T (Trm) cells accumulated in cecal lamina propria following E. falciformis infection in mice, especially after reinfection. In convalescent mice challenged with a second infection, E. falciformis burden diminished within 48-72 h. Deep-sequencing revealed that CD8+ Trm cells were characterized by rapid up-regulation of effector genes encoding pro-inflammatory cytokines and cytotoxic effector molecules. While FTY720 (Fingolimod) treatment prevented the trafficking of CD8+ T cells in peripheral circulation and exacerbated primary E. falciformis infection, such treatment had no impact on the expansion of CD8+ Trm cells in convalescent mice receiving secondary infection. Adoptive transfer of cecal CD8+ Trm cells conferred immune protection in naïve mice, indicating that these cells provide direct and effective protection against infection. Overall, our findings not only explain a protective mechanism of live oocyst-based anti-Eimeria vaccines but also provide a valuable correlate for assessing vaccines against other protozoan diseases.
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