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A technique for improved cryopreservation at -196 degrees C of Brugia spp. microfilariae has been developed by modifications of a procedure previously used with Onchocerca spp. A double incubation in ethanediol (ED) solutions, firstly at 37 degrees C in 10% (v/v) ED for 15 min and secondly at 0 degrees C in 40% (v/v) ED for 45 sec followed by plunging into liquid nitrogen, resulted in over 90% of the microfilariae of B. malayi exhibiting normal motility. When used with B. pahangi microfilariae, followed by blood-feeding to Aedes aegypti (Bels New strain) a third-stage larvae yield of 4.04 per fly was obtained, compared with 5.16 in the unfrozen group. This represents an infectivity level of 78% compared with unfrozen controls. The technique also has the benefits of being suitable for field conditions, requiring no sophisticated equipment, merely some ice, ethanediol and liquid nitrogen.

A range of culture conditions were examined to optimize parasite maintenance. Using male worms in a cell-free system, good results were obtained with medium NCTC 135 + 10% inactivated calf serum (IFCS) in an atmosphere of 95% N2/5% CO2 (median survival time 45 days). Survival was increased to 6-7 months using medium MEM + 10% IFCS + LLCMK2 (monkey kidney) feeder cells in a gas phase of 5% CO2 in air. Worms exposed to collagenase solution (5 mg/ml) were subsequently less motile and survived shorter periods compared to unexposed controls. The drug responses of worms (in vitro) were examined using 13 antiparasitic compounds. Ivermectin and CGP 6140 were among the most active, with the majority of drugs significantly affecting motility levels at a concentration of 5 x 10(-5) M or less. This system may provide useful information on the intrinsic activity of new compounds. A technique was developed for the successful cryopreservation of males in liquid nitrogen using ethanediol as a cryoprotectant in a 2-step incubation procedure, thereby enabling the long-term storage and transportation of worms. In conclusion, the common bovine parasite O. gutturosa provides a practical alternative for research in the absence of O. volvulus.

Experimental describe the use of ethanediol to store Onchocerca microfilariae in liquid nitrogen. The technique involves a 2-stage incubation of the parasites at 37 degrees C and 0 degrees C in ethanediol, before rapid cooling to -196 degrees C. Viability has been assessed by motility, by migration in a proxy host and by development to the infective stage in the insect vector. A total of 71-79% of the cryopreserved microfilariae was shown to be viable compared to unfrozen controls. The technique is simple, inexpensive and very effective when compared to previous cryopreservation procedures for microfilariae and should be particularly suited for use under field conditions.

Experiments involved the use of methanol and ethanediol (ED) as potential cryoprotectants for the storage of adult male Onchocerca gutturosa at low temperatures. Worms were incubated with or without 20% (v/v) methanol for 15 min at 0 degrees C before cooling to -10 degrees, -25 degrees or -79 degrees C. Results of trials of storage from 1 to 14 days showed that prolonged survival could only be obtained by storing at -79 degrees C without the addition of cryoprotectant. However, worms were also successfully cryopreserved in liquid nitrogen (-196 degrees C) using a simple 2-step procedure in which ED was used as an essential cryoprotectant. This schedule involved an initial incubation of parasites in serum free media with 10% (v/v) ED at 37 degrees C for 15 min, followed by incubation in 40% (v/v) ED for 10 min at 0 degrees C on crushed ice. Worms were then transferred individually into 20 microliter drops of 40% (v/v) ethanediol on glass coverslips at 0 degrees C and immediately plunged into liquid nitrogen. A rapid thaw of the parasites resulted in normal motility and survival levels equivalent to unfrozen controls. This technique will be useful for the long-term storage and transportation of this and possibly other species of Onchocerca parasites.