qPCR analysis of TsSP transcription level at different stages of T. spiralis. The TsSP mRNA from AW at 3 and 6 dpi, and NBL, ML and IIL were amplified by qPCR. The TsSP mRNA transcription levels were calculated via the Ct (2−ΔΔCt) method. G3PDH was utilized as a housekeeping gene control. TsSP transcription level was not statistically different among different stages of T. spiralis (P = 0.05).