Title

Investigation of the requirements for development of Brugia malayi infective stage larvae

Date of Completion

January 2002

Keywords

Biology, Microbiology

Degree

Ph.D.

Abstract

Brugia malayi is a filarial nematode and one of the causative agents of lymphatic filariasis, a disease afflicting an estimated 120 million people worldwide. Despite its clinical importance, little is known about the biology of the parasite itself. In order to gain a better understanding of the development of the infective stage (L3) larvae within the vertebrate host, we developed a serum-free system capable of supporting maturation of B. malayi L3 larvae to the L4 stage in vitro. This system consisted of standard cell-culture medium supplemented with polyunsaturated fatty acids (linoleic, linolenic, or arachidonic acids) and the basidiomycetous yeast Rhodotorula minuta. This system is substantially more defined than previously system and more easily reproducible. The L4 larvae generated are viable and capable of further development in a permissive host. Using this system as a tool, we were able to investigate the requirements for development of the L3 larvae. We found that larvae were benefiting from an unstable, secreted product of R. minuta and that this product was crucial during days 4 and 5 of culture. The requirement for polyunsaturated fatty acids for successful molting led us to investigate a possible role for eicosanoid products in L3 development. We found inhibitors of the lipoxygenase arm of the pathway, but not the cyclooxygenase arm, blocked development. Furthermore, inhibitors of enzymes and receptors operating downstream of lipoxygenase were similarly able to block maturation of L3 larvae. When tested on the related filarial parasite, Dirofilaria immitis, a subset of these inhibitors was capable of disrupting development. Finally, the culture system provided us with the opportunity to evaluate a recent concept in filarial research—the role of the rickettsia-like endosymbiont Wolbachia in filarial development. We found the anti-rickettsial drug tetracycline completely blocked L3 development. Overall, these studies have identified several previously unappreciated pathways involved in L3 development. These results highlight potential areas of vulnerability that may be exploited for the development of chemotherapeutic and immunoprophylactic interventions against B. malayi in the future. ^