Figure 3

Differential expression and localisation of E. tenella oocyst wall proteins. (a) (d) Quantitative reverse-transcriptase PCR was carried out on different developmental stages of E. tenella, including merozoites (M), gametocytes (G), unsporulated (U) and sporulated oocysts (S). The relative transcript abundance of etowp6 (a) and ethowp1 (d) was determined relative to et18S small subunit ribosomal RNA for each developmental stage. **** indicates a statistically significant difference of P < 0.001 between one sample and all other samples. (b) (e) Different developmental stages of E. tenella (M, G, U and S), as well as an uninfected host control (−) were analysed by Western blot using anti-EtOWP6 rabbit sera (diluted 1 in 1,000) (b) and anti-EtHOWP1 mouse sera (1 in 4,000) (e). Molecular weight marker is listed to the left in kDa. (c) (f) (g) Sections of E. tenella-infected chicken caeca (144 h post-infection) were analysed by immunofluorescence microscopy with DAPI counterstaining. (c) Anti-EtOWP6 rabbit sera (1 in 200) localises to wall-forming bodies type I (WFBI) while anti-GAM56 mouse sera (1 in 1,000) localises to wall-forming bodies type II (WFBII). Scale bar = 10 μm. (f) Anti-EtHOWP1 mouse sera (1 in 2,000) and anti-GAM56 rabbit sera (1 in 1,000) co-localise to WFBII. Scale bar = 10 μm. (g) Confocal imaging of anti-EtHOWP1 rabbit sera (1 in 2,000) demonstrates WFBII staining, while WFBI remain unstained. DAPI stains mid-stage microgametes (MMi). Scale bar = 2 μm. (h) Broken and intact E. tenella unsporulated oocysts were probed with anti-EtHOWP1 (1 in 2,000). Oocyst walls fluoresce blue under UV excitation, due to the presence of cross-linked dityrosine. An overlay of bright field (BF) and anti-EtHOWP1 staining shows localisation to the walls of broken oocysts (the inner layer). Scale bar = 10 μm.