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

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.