Transfected Eimeria tenella Could Complete Its Endogenous Development In Vitro

Abstract

Live attenuated coccidiosis vaccines could be used as powerful carriers, expressing exogenous viral and bacterial antigens, to induce protective immunity against pathogenic organisms. We investigated the ability of Eimeria tenella to express an exogenous gene in vitro. Eimeria tenella sporozoites were transfected with the plasmid pH4-2EYFP-Actin3 containing the yellow fluorescent protein gene (yfp) and inoculated into primary chicken kidney cells (PCKCs), followed by incubation at 41 C in a 5% CO2 chamber. Fluorescent sporozoites were observed as early as 15-20 hr post-inoculation (PI). Fluorescence displayed by the expressed YFP protein was visible throughout the schizogony and gametogony stages of the tranfected E. tenella. Fluorescent oocysts were found between 200-327 hr PI. Higher fluorescence intensity was observed in the nucleus than in other compartments of the transfectants, while little or no fluorescence was seen in the refractile globule. The diversity of schizonts, particularly of the first generation, was presented by fluorescent nuclei arranged in different patterns. Our results demonstrated the ability of E. tenella to express an exogenous gene throughout the endogenous development in vitro. Completion of the endogenous development of transfected E. tenella in cell cultures will facilitate the study of foreign antigen expression in Eimeria spp., paving the way for the development of an Eimeria spp. vector vaccine that also carries and delivers other vaccines by oral administration.

piggyBac Transposon-Mediated Transgenesis in the Apicomplexan Parasite Eimeria tenella

“…2G ) . As described previously [19], most of the fluorescent protein was targeted to the parasite nucleus under the influence of the 90-bp NLS. Transfection with circular donor plasmid pHEA-Bac and the restriction enzyme Asc I resulted in fluorescent parasites in the in vitro cultures at a similar level to the cultures infected with co-transfected (pHEA-Bac + pH 4-IFP2-A + Asc I ) parasites (Fig.…”

“…Freshly purified sporozoites were washed once in incomplete cytomix (10 mM K2HPO4:KH2PO4, pH 7.6; 120 mM KCl; 0.15 mM CaCl2; 25 mM HEPES; 2 mM EGTA; and 5 mM MgCl2) [6] and then suspended in the same buffer, supplemented with 2 mM ATP and 5 mM glutathione. In vitro cell culture of transfectants was carried out using primary chicken kidney cells (PCKCs) prepared from a ten-day-old chicken as described previously [19]. Arbor Acres (AA) broiler chickens were used for preparation of PCKCs and parasite propagation in vivo.…”

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

“…Eimerian parasites can stimulate strong antibody and cell-mediated immunity ( Wallach, 2010 ). Their exceptional potential as vectors delivering heterologous antigens to the mucosal immune system were well recognized ( Shi et al, 2008 ; Yan et al, 2009 ; Clark et al, 2012 ). Two transgenic lines of E. tenella were reported to induce exogenous antigen-specific immune responses ( Huang et al, 2011 ; Clark et al, 2012 ), and another transgenic E. mitis expressing chicken interleukin 2 strongly enhanced cellular immunity against wild type parasite challenge ( Li et al, 2015b ).…”

Development ofEimeria nieschulzi(Coccidia, Apicomplexa) Gamonts and Oocysts in Primary Fetal Rat Cells

“…Our observation in small bowel smears showed only weak fluorescence signals in microgametes (not shown) and no micrographs could be taken through the rapid movement of the microgametes. Shi et al [24] described the development of yfp-expressing microgametes of Eimeria tenella in vitro, but did not show any transmitted light micrographs. They mentioned that the nuclei in microgametes in immature microgamonts were not clear.…”