Arkadi Kundik (Berlin / DE), Univ.-Prof. Dr. rer. nat. Susanne Hartmann (Berlin / DE), Prof. Dr. Friederike Ebner (Berlin / DE)
Abstract text
Helminth infections are a major health burden of humans and animals alike. Due to rising resistances against anthelmintics, there is an urgent need for new drugs. Current approaches for anthelmintic drug screening are considered subjective, laborious, and low in throughput since their endpoint readouts have to be evaluated microscopically. Here, we aimed to establish and optimize a fluorometric-based assay using resazurin and evaluated the assay by studying the metabolic activity of Ascaris suum (A. suum) larvae (L3), a highly prevalent parasite in swine.
A. suum L3 were assessed for their potential to reduce the non-fluorescent resazurin to the highly fluorescent resorufin using up to 1000 L3 and resazurin concentrations up to 15µg/ml. The assay was carried out in 96-microwell plates and fluorescence intensities were measured 24h post-incubation with the dye. Additionally, fluorescence microscopy was used to assess the resazurin reduction site within larvae. Finally, the assay was evaluated with larvae that have been exposed to different metabolic stressors or anthelmintics.
Our data show that resazurin is reduced in vital A. suum L3 to resorufin and released into the media. An intact L2 sheath around the L3 of A. suum completely prevented the uptake of resazurin while in unsheathed L3 a fluorescence signal was detected along the intestine. L3 which were metabolically affected by methanol or heat exposure showed a gradually decreased resazurin reduction activity. Exposure to ivermectin at 0.625µM, mebendazole at 5µM and thiabendazole from 10µM to 100µM for 24h significantly decreased larval resazurin reduction activity by 55%, 73% and 70% to 89%, respectively.
Together, our data show that metabolic stressors or anthelmintic drugs significantly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy to use high-throughput method to measure larval metabolic activity in vitro.