Development of a serum-free system for the in vitro cultivation of Brugia malayi infective-stage larvae

Over the past several years, numerous attempts have been made to culture th e infective-stage (L3) larvae of the human filarial parasite Brugia malayi in an in vitro system that promotes molting to the fourth larval stage (L4) . Although there have been reports in the literature of successful L3 to L4 development in vitro, all of these systems have required serum supplementa tion. The complexity of serum as a culture supplement has made reproducibil ity of results and identification of specific factors necessary for L3 deve lopment problematic. We have developed a serum-free in vitro system consist ing of RPMI 1640 supplemented with one of three fatty acids (arachidonic, l inoleic, or linolenic) that supports consistent and reproducible molting to the fourth larval stage in the presence of a basidiomycetous yeast, Rhodot orula minuta. Coculture with this yeast, initially isolated as a culture co ntaminant, is required for successful molting. In serum-free cultures lacki ng R. minuta, L3 larvae survive for upward of 2 weeks, but do not molt succ essfully. The L4 larvae generated in cultures containing R. minuta are well formed, as seen by light and electron microscopy, and are capable of furth er development upon transfer to a permissive host. This culture system is i nexpensive and easily reproducible, thus making it a useful tool for studyi ng the requirements for the development of B. malayi L3. (C) 2000 Academic Press.