The exsheathment of Brugia pahangi microfilariae under controlled conditions in vitro

Two reproducible techniques for the exsheathment in vitro of microfilariae of Brugia pahangi, and other sheathed microfilariae, are described. Microfilariae were isolated from infected cat blood by filtration and suspended in Hank's Balanced Salt Solution. The first technique involved the incubation of isolated microfilariae for one hour in 20 mM CaCl2 in a phosphate-free Balanced Salt Solution, during which time approximately 90% of the microfilariae lost their sheaths. The second method of exsheathing microfilariae of B. pahangi involved exposure of microfilariae to solutions of endopeptidase (5.8 units/ml) or papaya extract protease (3.0 units/ml) in Ca2+-free HBSS. Exsheathment rates of 95--100% occurred within 30 minutes in both enzyme solutions. Both the Ca2+ ion and the endopeptidase technique have proven equally effective in stimulating exsheathment of microfilariae of Brugia malayi, Wuchereria bancrofti and Litomosoides carinii. Such artificially exsheathed microfilariae are used for in vitro cultivation studies. The viability of Ca2+- and endopeptidase-exsheathed microfilariae of B. pahangi has been confirmed by inoculation of exsheathed larvae into susceptible female mosquitoes.