An in vitro model for studying gene expression in Parascaris univalens larvae

Abstract: Parascaris univalens, equine roundworm, is a large nematode infecting young horses and causes respiratory syndromes, impaired condition and reduced growth. Adults can reach up to 50 cm in length and heavy infection can lead to obstruction and rupture of the small intestines due to the worm’s size. Emerging resistance against all substances available for treatment is threatening animal welfare. Research in the area is troubled by the limited accessibility, size and poor in vitro survival of adult worms. To increase understanding of genes involved in resistance, this study focused on genes involved in drug metabolism and had three aims: i) Develop an in vitro model for P. univalens L3 larvae, ii) compare gene expression between larvae and adult, and iii) investigate how drug exposure affects gene expression in larvae. Larvae was hatched from eggs collected from faecal samples, cultured and exposed to ivermectin. The expression of orthologous genes of Caenorhabditis elegans CYP-14A2, GST-20 and SKN-1, Haemonchus contortus UGT and Ixodes scapularis putative ABC-transporter was compared between unexposed larvae and an adult worm through qPCR. CYP-14A2, GST-20, SKN-1 and the putative ABC-transporter were further investigated through qPCR in larvae exposed to 10−15 M or 10−7 M ivermectin. The model was functional, with a hatching ratio of 50% and 85% larval survival after 13 days culture in RPMI medium. Differential gene expression between larvae and adult was detected for CYP-14A2, ABC-transporter, UGT and GST-20 orthologs. Trends of down regulation in ivermectin- exposed larvae were detected for the ABC-transporter and CYP-14A2 orthologs. Further research could highlight significant differences both between life stages and in larvae after drug exposure.