Authors

Discussion

Background: The recent increase in anthelmintic resistance of parasitic nematodes, for example in the equine roundworm Parascaris univalens, calls for further research to understand the complex interactions between host and parasite. Although host immune modulation via parasite excretory-secretory products have been described in different experimental models, most host-parasite species-specific and zoonotic interactions remain unexplored. This is largely due to the complex and species-specific infective life cycle and a lack of proper in vitro models to effectively test these interactions.

In vitro model: To this end, we have begun investigating the feasibility of cell culture models during interactions with the infective L3 stages of Toxocara canis and Parascaris univalens. To help quantify our methods, we used the resazurin-resorufin assay. Resazurin is a low molecular weight, non-toxic dye, which during normal metabolism in the cell is non-reversibly reduced to resorufin. Under excitation, resorufin produces a distinct red fluorescence signature, giving a direct readout of metabolic levels. The resazurin-resorufin assay gives robust and repeatable results in a variety of cell types and under a variety of cellular stressors. In addition to metabolic readout, thrashing assays via the WormAssay program, were also performed to judge the effect of stressors on the L3 larva in a non-biased way. Our main stressor was thiabendazol, an actively used anthelmintic of the benzimidazole family know to selectively target beta-tubulin.

Results: When infective L3 larva of Toxocara canis or Parascaris univalens were co-cultured for 48-hours with adherent human intestinal epithelial cells (Caco-2) we have seen significant changes in overall cellular metabolism (change in resorufin fluorescence), in comparison to control incubations with either L3 larva or adherent cells. When in contact with host cells a significant decrease in thrashing rates in Toxocara canis L3 larva, as well as a shift in thrashing behavior was also observed. Furthermore, we observed a significant decrease in overall metabolic rate in co-cultures of Toxocara canis and Caco-2 when exposed for 48-hours to 100µM thiabendazol, a decrease that was not seen in control Caco-2 cells when they were cultured alone under the same thiabendazol concentration. Our results indicate that the early phase in vitro co-culture model is resulting in metabolic changes for both host and parasite and parasite behavioral changes which indicates a potential model for further research.