Molecular Engineering of a Dual-Cassette Transgenic Eimeria mitis Expressing Chicken IL-4 and Nuclear-Localized EYFP

Genetic manipulation of Eimeria species has emerged as a pivotal strategy for developing next-generation live vaccines capable of delivering immunomodulatory molecules directly to the host intestinal mucosa. In this study, a dual-cassette expression plasmid was constructed to generate a transgenic line of Eimeria mitis (E. mitis) capable of expressing chicken interleukin-4 (ChIL-4) and enhanced yellow fluorescent protein (EYFP). The ChIL-4 open reading frame was amplified from chicken spleen lymphocyte cDNA, while DHFR-TSm2m3 and EYFP fragments were obtained from previously established constructs to assemble a fused DHFR-EYFP reporter cassette. The final double-cassette plasmid (pHDEAAssIL4A) contained regulatory elements derived from Eimeria tenella and a secretion signal to ensure extracellular IL-4 release. Transfection of E. mitis sporozoites was performed using nucleofection under REMI conditions with either SnaBI-linearized or circular plasmid DNA. Linear DNA yielded markedly superior transfection efficiency and fluorescence intensity, whereas circular plasmid produced negligible early transgenic oocyst output and weaker fluorescence, consistent with prior observations in Eimeria species. Stable EYFP-expressing E. mitis populations were enriched through successive in vivo passages under pyrimethamine selection and FACS sorting, ultimately reaching over 90% fluorescence-positive sporulated oocysts by the seventh passage for the linear-plasmid group. Molecular verification confirmed successful chromosomal integration of both EYFP and ChIL-4. PCR analysis consistently detected the expected 411 bp IL-4 fragment and 726 bp EYFP fragment in transgenic genomic DNA, whereas these amplicons were absent in wild-type controls. These results validate the fact that a double-cassette vector system can be used to successfully transfect E. mitis, and express EYFP and IL-4 across generations in a stable manner. This is because the effective production of this IL-4-expressing line can be a promising step to the use of E. mitis as a genetically engineered vaccine carrier and the investigation of the IL-4-dependent suppression of host immunity.