Quantifying metabolic activity of Ascaris suum L3 using resazurin reduction.
Anthelmintics
Ascaris suum
Drug screening assay
Larvae
Metabolic activity
Resazurin
Viability assay
Journal
Parasites & vectors
ISSN: 1756-3305
Titre abrégé: Parasit Vectors
Pays: England
ID NLM: 101462774
Informations de publication
Date de publication:
19 Jul 2023
19 Jul 2023
Historique:
received:
25
01
2023
accepted:
04
07
2023
medline:
21
7
2023
pubmed:
20
7
2023
entrez:
19
7
2023
Statut:
epublish
Résumé
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. 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. 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% CO 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.
Sections du résumé
BACKGROUND
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
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
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% CO
CONCLUSIONS
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.
Identifiants
pubmed: 37468906
doi: 10.1186/s13071-023-05871-5
pii: 10.1186/s13071-023-05871-5
pmc: PMC10357624
doi:
Substances chimiques
resazurin
1FN9YD6968
Methanol
Y4S76JWI15
Anthelmintics
0
Xanthenes
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
243Subventions
Organisme : Deutsche Forschungsgemeinschaft
ID : Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 251133687
Informations de copyright
© 2023. The Author(s).
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