Quantifying metabolic activity of Ascaris suum L3 using resazurin reduction

Abstract

Background: Helminth infections are an important public health problem in humans and have an even greater impact on domestic animal and livestock welfare. Current readouts for anthelmintic drug screening assays are stage development, migration, or motility that can be subjective, laborious, and low in throughput. The aim of this study was to apply and optimize a fluorometric technique using resazurin for evaluating changes in the metabolic activity of Ascaris suum third-stage larvae (L3), a parasite of high economic relevance in swine.

Methods: Ascaris suum L3 were mechanically hatched from 6- to 8-week embryonated and sucrose-gradient-enriched eggs. Resazurin dye and A. suum L3 were titrated in 96-well microtiter plates, and resazurin reduction activity was assessed by fluorometry after 24 h of incubation. Fluorescence microscopy was used to localize the resazurin reduction site within the larvae. Finally, we exposed A. suum L3 to various stress conditions including heat, methanol, and anthelmintics, and investigated their impact on larval metabolism through resazurin reduction activity.

Results: We show that the non-fluorescent dye resazurin is reduced inside vital A. suum L3 to fluorescent resorufin and released into the culture media. Optimal assay parameters are 100-1000 L3 per well, a resazurin concentration of 7.5 µg/ml, and incubation at 37 °C/5% CO₂ for 24 h. An intact L2 sheath around the L3 of A. suum completely prevents the uptake of resazurin, while in unsheathed L3, the most intense fluorescence signal is observed along the larval midgut. L3 exposed to methanol or heat show a gradually decreased resazurin reduction activity. In addition, 24 h exposure to ivermectin at 0.625 µM, mebendazole at 5 µM, and thiabendazole from 10 to 100 µM significantly decreased larval metabolic activity by 55%, 73%, and 70% to 89%, respectively.

Conclusions: Together, our results show that both metabolic stressors and anthelmintic drugs significantly and reproducibly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy-to-use method to evaluate metabolic activity of A. suum L3 in vitro.

Keywords: Anthelmintics; Ascaris suum; Drug screening assay; Larvae; Metabolic activity; Resazurin; Viability assay.

Fig. 1

Purification of embryonated A. suum eggs using sucrose density gradient centrifugation. a Exemplary flow cytometric scatter plots depicting forward (FSC-A) and sideward scatter (SSC-A) properties of un-embryonated A. suum eggs in suspension before embryonation (left) and a mixed egg suspension after 6 weeks of embryonation (right). b Histograms of weekly FSC/SSC measurements of A. suum eggs from week 0 to week 6 after start of embryonation. c Workflow for separating fully embryonated A. suum eggs from undeveloped eggs. The uterine layer is removed prior to gradient purification. Mixed egg suspensions are separated using four sucrose layers—18% (w/v), 20%, 23%, and 25%—and eggs are evaluated microscopically (black scale bar: 50 µm) for each layer

Fig. 2

Ascaris suum L3 reduce the metabolic indicator dye resazurin. a Biochemical reaction in which the non-fluorescent dye resazurin (blue) is reduced to fluorescent resorufin (pink). The reaction requires the presence of diaphorase enzymes and NADH/H+. b Fluorescence emission spectra of resazurin (blue dashed curve) and resorufin (pink solid curve) in HBSS-AB at λex = 540 nm. c Titration of resazurin from 0 to 15 µg/ml with a fixed number of 500 in vitro-hatched A. suum larvae per well shows a hyperbolic relationship (df = 7, R² = 0.98, best-fit ½max = 1.6 µg/ml). d Titration of A. suum L3 numbers from 0 to 1000 in vitro-hatched L3 with the fixed concentration of 7.5 µg/ml resazurin reveals a strong linear correlation (R² = 0.99, equation: Y = 17.89x + 122.3, r = 0.99, with P < 0.0001). c, d Fluorescence intensity measured at λem = 590 nm and λex = 540 nm. Black dots represent fluorescence intensity from single wells (n = 3 technical replicates). Grey curve represents the curve of best fit (least-squares method)

Fig. 3

Ascaris suum L3 reduce resazurin to resorufin and release the majority into the media. a Comparison of fluorescence intensity before and after larval removal: 500 A. suum L3 per well were measured after incubation with 7.5 µg/ml resazurin for 24 h (+ larvae) and after removal of L3 (− larvae). Both groups (n = 12 technical replicates) were compared using the paired t-test (t = 3.774, df = 11, P = 0.0031). b Excretory–secretory (ES) products of A. suum L3 do not convert resazurin into resorufin. Fluorescence intensity of ES-containing supernatants collected from 62, 125, 250, and 500 L3/well after incubation with 7.5 µg/ml resazurin for 24 h. Scatter plot shows mean and standard deviation (SD) (solid grey line) of relative fluorescence units (RFUs), black dots represent RFUs of single wells, and the dotted line represents y = 0; n = 3 technical replicates

Fig. 4

Resorufin localization inside A. suum L3. a Experimental set-up for localization of resorufin inside living larvae by fluorescence microscopy. b Control larvae incubated without resazurin. c Image acquisition 3 h after addition of 7.5 µg/ml resazurin. Bright field (left), fluorescence (middle), and overlay (right) images of resorufin inside unsheathed L3. A magnified view of the posterior and anterior end of a non-fluorescent larva depicting the L2 cuticle around the L3 (black arrow heads). Image acquisition settings: λex = 555 nm with a bandpass filter for λem = 579–604 nm. Black scale bar: 100 µm, yellow scale bar: 40 µm, white scale bar: 20 µm

Fig. 5

Resazurin reduction assay enables quantification of metabolic impairment. Metabolic activity was assessed for A. suum larvae after a heat treatment at 60 °C for 0 to 8 min and b exposure to different methanol concentrations for 3 h using the resazurin reduction assay. Scatter plots represent mean (SD) fluorescence measurements of n = 3 technical replicates per treatment. Asterisks indicate statistical significance by Dunnett’s test: *P < 0.05, **P < 0.01, ****P < 0.0001; ns: not statistically significant

Fig. 6

Resazurin reduction assay in drug testing. Larvae were exposed to anthelmintics for 24 h, following an incubation with 7.5 µg/ml resazurin for 24 h. The relative fluorescence intensities were measured fluorometrically for a DMSO-treated larvae from 0 to 4 vol%. b Thiabendazole in 0.5% DMSO applied from 0 to 100 µM. c Ivermectin in 0.5% DMSO applied at 625 nM. d Mebendazole in 0.5% DMSO applied at 5 µM. Scatter plots represent the mean fluorescence intensity (λex = 540, λem = 590) for n = 3 technical replicates. Asterisks indicate statistical significance by Dunnett’s test for DMSO and thiabendazole and by t-test for ivermectin and mebendazole: *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001; ns: not statistically significant. All drug assays were independently repeated using in vitro-hatched A. suum L3 from different batches