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Survival and infectivity of Brugia malayi microfilariae after cryopreservation
1Guizhou Provincial Institute of Parasitic Diseases, Guiyang, People's Republic of China.
Abstract:
Methods were studied for the cryopreservation of microfilariae of periodic Brugia malayi. RPMI-1640 tissue culture medium containing 6% dimethyl sulfoxide (DMSO) and 15% newborn calf serum was used as cryoprotectant. Samples were frozen slowly in the vapor phase of liquid nitrogen prior to emersion in liquid nitrogen (-196 degrees C). The freezing rate was -0.5 to -1.0 degrees C per minute, microfilariae remained viable for as long as, 212 and 375 days, survival rates were 94 to 98% and they were infective to Aedes togoi mosquitos. The infective larvae (L3) were obtained for 10-11 days after feeding at 28 degrees C room-temperature and the infection rate of L3 in test mosquitos was 22.4-30.6%. All DMSO should be removed from the freezing medium to restore microfilariae activity after freezing.
Insights
Cryopreservation of Brugia malayi microfilariae using dimethyl sulfoxide (DMSO) and newborn calf serum achieved high survival rates up to 375 days. These cryopreserved microfilariae remained infective to mosquitoes, demonstrating successful long-term storage.
Area of Science:
- Parasitology
- Cryobiology
- Molecular Biology
Background:
- Brugia malayi is a filarial nematode responsible for lymphatic filariasis.
- Effective cryopreservation methods are crucial for maintaining parasite stocks for research and drug development.
Purpose of the Study:
- To establish and optimize methods for the cryopreservation of Brugia malayi microfilariae.
- To assess the viability and infectivity of cryopreserved microfilariae.
Main Methods:
- Microfilariae were cryopreserved using RPMI-1640 medium with 6% dimethyl sulfoxide (DMSO) and 15% newborn calf serum.
- Slow freezing was performed in the vapor phase of liquid nitrogen, followed by immersion at -196°C.
- Viability and infectivity assays were conducted after various storage durations.
Main Results:
- High survival rates (94-98%) were observed for microfilariae stored for up to 375 days.
- Cryopreserved microfilariae successfully developed into infective larvae (L3) after feeding to Aedes togoi mosquitoes.
- Infection rates of L3 in mosquitoes ranged from 22.4-30.6%.
Conclusions:
- Cryopreservation of Brugia malayi microfilariae is feasible and maintains viability and infectivity.
- DMSO removal post-thawing is essential for restoring microfilariae activity.
- This method supports long-term storage of Brugia malayi for research purposes.