Expression and neutralization of CpLTL in oocysts and developmental stages in C. parvum. (A) Expression of CpLTL in oocysts (first panel), excystation (second panel), sporozoites (third panel), oocyst wall (fourth panel), and intracellular developmental stages of C. parvum in HCT-8 cell cultures at 24 (fifth panel) and 48 h (sixth panel). The images were taken under differential interference contrast (DIC), with nuclei being counter-stained with 4′, 6-diamidino-2-phenylindole (DAPI), parasites stained by immunofluorescence with Alexa Fluor^® 594-labelled secondary antibody, and superimposition of the three images (Merged). Bars = 5 μm. (B) Expression level of the CpLTL gene at various C. parvum culture times as determined by qRT-PCR. Data from the Cryptosporidium 18S rRNA gene were used as an internal control for data normalization. The CpLDH gene was detected in parallel as a reference and for quality control. Data are presented as Mean ± SD from three replicate cultivation assays. (C) Neutralization efficiency of polyclonal antibodies against CpLTL in C. parvum culture. Oocysts were pre-incubated in medium with 1:500, 1:100, and 1:50 dilutions of pre-immune serum and polyclonal antibodies, with medium alone as a control. Data are presented as Mean ± SD from three replicate assays (p < 0.05).