Establishment of an in vitro culture system to study the developmental biology (growth, mating and nodule formation) of Onchocerca volvulus with implications for anti-onchocerca drug discovery and screening

Abstract

Background Infections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after two decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology, especially mating behaviour and nodule formation, as well as development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite’s reproductive and behavioural pattern.Methodology/Principal Findings Prior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium 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 supported the viability and motility of O. volvulus larvae for up to 84 days (DMEM–10%NCS), while the co-culture system (DMEM–10%FBS–LLC-MK2) extended the worm survival period to 315 days. Co-culture systems alone promoted the two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates observed in DMEM–10%FBS–LLC-MK2 (69.2±30 %), while no moult was observed in DMEM–10%NCS–LEC condition. O. volvulus adult worms mating and even mating competitions were observed in DMEM–10% FBS –LLC-MK2 co-culture system. Early nodulogenesis was observed in both DMEM–10% FBS–LLC-MK2 and DMEM– 10%NCS–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 reproductive adult stages. Thus, this platform gives potential for the investigation of mating, mating competition and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for onchocercacidal drug screening.Author summary River blindness affects people living in mostly remote and underserved rural communities in some of the poorest areas of the world. Although significant efforts have been achieved towards the reduction of disease morbidity, onchocerciasis still affect million of people in Sub-Saharan Africa. The current control strategy is the annual mass administration of ivermectin which have accumulated several drawbacks overtime: as the sole microfilaricidal action of the drug, very long treatment period (15-17 years) and reports of ivermectin losing its efficacy; Therefore, raising the urgent need for new onchocercacidal molecules. Our study has established an in vitro platform capable of supporting the growth and development of all developmental stages of O. volvulus (L3 infective stage, L4, L5 and adult worms), moreover the platform provided more insight on O. volvulus adult worms reproductive and behavioural pattern. Our findings provide more avenues for mass production of different parasite stages, the investigation of parasite developmental biology and the identification of targets for drug discovery against different phases of development of this filaria parasiteAbbreviationsAPOCAfrican Programme for Onchocerciasis ControlOCPOnchocerciasis Control ProgrammePTSPost-Treatment SurveillanceHC-04Human Hepatocyte cellsHEK-293embryonic human kidneys cellsLLC-MK2Monkey Kidney cellsLECMouse embryonic lung cellsL3Infective LarvaeL4Stage 4 larvaeL5Stage 5 larvaeRPMIRoswell Park Memorial InstituteDMEMDulbecco’s Minimum Essential MediumMEMMinimum Essential MediumIMDMIscove’s Modified Dulbecco MediumNCTCNew jersey Cell Type CollectionNCSNew-born Calf SerumFBSFoetal Bovine SerumBCSBovine Calf SerumMDAMass Drug AdministrationWHOWorld Health OrganisationCMFLCommunity Microfilariae LoadsEGFEpidermal Growth FactorTGF-αTransforming Growth Factor - alphaIGF - IInsulin-like Growth Factor – IPDGFPalettes-Derived Growth FactorDDTDichlorodiphenyltrichloroethane.View Full Text