Establishment of an in vitro culture system to study the developmental biology of Onchocerca volvulus with implications for anti-Onchocerca drug discovery and screening.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101291488 Publication Model: eCollection Cited Medium: Internet ISSN: 1935-2735 (Electronic) Linking ISSN: 19352727 NLM ISO Abbreviation: PLoS Negl Trop Dis Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Larva/*growth & development ; Onchocerca volvulus/*growth & development ; Parasitic Sensitivity Tests/*methods; Animals ; Culture Media/chemistry ; Developmental Biology ; Drug Evaluation, Preclinical/methods ; Feeder Cells/physiology ; Female ; Larva/physiology ; Male ; Molting ; Onchocerca volvulus/physiology

Background: Infections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour.
Methodology/principal Findings: Prior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2-3 days. The cell-free culture system (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK2 and DMEM supplemented with 10% NCS and seeded on LLC-MK2 systems.
Conclusions/significance: The present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening.
Competing Interests: None

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0 (Culture Media)

Date Created: 20210209 Date Completed: 20210608 Latest Revision: 20210608

20240104

PMC7899360

10.1371/journal.pntd.0008513

33561123