The effects of doxycycline on brugia malayi microfilariae

Abstract

Lymphatic filariasis, is a mosquito-borne disease, caused by Wuchereria bancrofti and Brugia malayi. After mating, fertile adult female worms will release an abundance of offspring (microfilariae; mf) into the host blood circulatory system. Transmissionblocking agents such as ivermectin, diethylcarbamazine, albendazole, as well as antirickettsial agents (e.g. doxycycline, rifampicin, and ciprofloxacin) have been used to reduce microfilarial density in human and animal reservoir hosts, to prevent disease transmission. Anti-rickettsial drugs also have the effect on the obligate intracellular gramnegative bacteria, Wolbachia, the mutualistic endosymbiont that appears to exert influence on filarial nematode embryonic and larval development, adult female fertility, and filarial survival. We investigated the effects of doxycycline, rifampicin and ciprofloxacin on B. malayi microfilarial motility, by using the minimum effective concentration (MEC). The minimum inhibitory concentration (MIC), the concentration of the anti-rickettsial drugs that could inhibit Wolbachia growth in mf, derived from the single copy gene ratio of Wolbachia versus nematode (wsp/hsp70) using the quantitative polymerase chain reaction (qPCR), was also demonstrated. Doxycycline was showed as the best effective antimicrobial agent. Doxycycline at 128 μg/ml (MEC) inhibited microfilarial motility completely at 12 h. Rifampicin and ciprofloxacin were less effective, both with MECs of >256 μg/ml at 12 h. Doxycycline MIC was 128 μg/ml, whereas rifampicin and ciprofloxacin MICs were >128 μg/ml, at 12 h. The MEC and MIC could be used to evaluate anti-Wolbachia or antifilarial agents for in vitro screening. To understand the molecular effect(s) of doxycycline on mf, we used microarray analysis to investigate temporal gene expression changes in B. malayi mf exposed in vitro to 20 μg/ml doxycycline as compared with non-treated control. By 61 h post-traetment, doxycycline-treated mf exhibited a significantly altered gene expression signature. We observed up-regulation of genes involved in protein folding such as, small heat shock protein and heat shock protein 90. In contrast, genes encoding for enzymes involved in the parasite mitochondrial electron transport chain, such as subunits of NADH dehydrogenase and cytochrome oxidase, were down-regulated. Our data suggest that doxycycline alters larval homeostasis either through a direct effect on the worm or through an indirect effect on the parasite’s endosymbiont Wolbachia.