Fig. 4

Relative gene expression and localization of the Eimeria tenella FOA1 and GAM-56 proteins in parasites grown in the chicken epithelial cell line, CLEC-213. a Relative gene expression of Etfoa1 and Etgam56 during parasite development stages. Left panel: RT-qPCR was performed on different enriched parasite stages of E. tenella: sporozoites (Sp) obtained by excystation, merozoites (M1) obtained from in vitro culture and gametes purified from caeca from infected chicken. Middle panel: RT-qPCR was performed on CLEC-213 infected with merozoites II from E. tenella wild type Wis strain (day 0 to day 2 p.i.). Right panel: RT-qPCR was carried out on CLEC-213 infected with sporozoites from the precocious strain of E. tenella, Wis-F-96 (day 1 to day 5 p.i.). The relative transcript expression of Etgam56 and Etfoa1 was defined relative to the E. tenella housekeeping gene, Et18S. Statistical analysis was performed using ANOVA followed by Dunn’s test. *P < 0.05 and **P < 0.01 indicate a statistically significant difference compared to previous free parasite stages (Sp and M1) or previous days of infection. Data are the mean ± SD of at least two biological replicates and three technical replicates. b Immunofluorescence analysis of microgametocytes and macrogametocytes production in CLEC-213 infected with merozoites II from E. tenella Wis strain (i, ii) or sporozoites from the precocious Wis-F-96 strain (iii, iv) was performed using anti-FOA1 mouse sera (i, iii) and anti-GAM56 rabbit sera (ii, iv) and the appropriate secondary antibodies (Alexa 594). Monolayers were mounted with DAPI counterstaining and parasites were visualised by fluorescent microscopy. Abbreviations: Sp, Sporozoites; MI, first generation merozoites; MII, second generation merozoites, Mi, microgametes (i, iii); Ma, macrogametes (ii, iv). Scale-bars: 5 μm