Onchocerca spp: Cryopreservation of microfilariae and subsequent development in the insect host

Abstract

Microfilariae of Onchocerca gutturosa, O. cervicalis and O. volvulus were successfully recovered after freezing, storage at −196 C, and thawing. The technique that produced maximum viability involved a two-step cooling schedule consisting of an initial slow cool of 1 C min −1 to an intermediate temperature of between −14 and −17 C, followed by a rapid cool into liquid nitrogen (taking about 1 sec). Upon rapid warming to 37 C, a high percentage of microfilariae showed normal motility. Following subcutaneous injection into T.O. mice, the microfilariae of O. gutturosa migrated to the skin of the ears and nose, and a proportion of them developed into third-stage larvae in the insect vector, Simulium ornatum. Microfilariae of O. volvulus also developed into third-stage larvae in this insect, while those of O. cervicalis developed similarly in their natural vector, Culicoides nubeculosus. This technique of preservation provides a good and reliable method for storage of viable microfilariae of these bovine, equine, and human Onchocerca spp.