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Concept: Brugia malayi

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Lymphatic filariasis (LF) is a leading cause of morbidity in the tropical world. It is caused by the filarial parasites Wuchereria bancrofti, Brugia malayi and Brugia timori and transmitted by vector mosquitoes. Currently a programme for the elimination of LF, Global Lympahtic Filariasis Elimination Programme (GPELF), is underway with the strategy of mass administration of single dose of diethylcarbamazine or ivermectin, in combination with an antihelminthic drug, albendazole. However, antifilarial drugs used in the progarmme are only microfilaricidal but not or only partially macrofilaricidal. Hence, there is a need to identify new targets for developing antifilarial drugs. Filarial parasites harbour rickettsial endosymbionts, Wolbachia sp., which play an important role in their biology and hence are considered as potential targets for antifilarial chemotherapy development. In this study, one of the cell division proteins of Wolbachia of the major lymphatic filarial parasite, Wuchereria bancrofti, viz., filamentation temperature-sensitive protein Z (FtsZ), was explored as a drug target. The gene coding for FtsZ protein was amplified from the genomic DNA of W. bancrofti, cloned and sequenced. The derived amino acid sequence of the gene revealed that FtsZ protein is 396 amino acids long and contained the tubulin motif (GGGTGTG) involved in GTP binding and the GTP hydrolyzing motif (NLDFAD). The FtsZ gene of endosymbiont showed limited sequence homology, but exhibited functional homology with β-tubulin of its host, W. bancrofti, as it had both the functional motifs and conserved amino acids that are critical for enzymatic activity. β-tubulin is the target for the anti-helminthic activity of albendazole and since FtsZ shares the functional homology with it may also be sensitive to albendazole. Therefore, the effect of albendazole was tested against Wolbachia occurring in mosquitoes instead of filarial parasites as the drug has lethal effect on the latter. Third instar larvae of Culex quinquefasciatus were treated with 0.25mg/ml of albendazole (test) or tetracycline (positive control) in the rearing medium for different intervals and tested for the presence of Wolbachia by FtsZ PCR. All the treated larvae were negative for the presence of the FtsZ band, whereas all the control larvae were positive. The findings of the study thus indicated that FtsZ is sensitive to albendazole. In view of this albendazole appears to have dual targets; FtsZ in Wolbachia and β- tubulin in W. bancrofti. Further, the functional domain of the gene was assessed for polymorphism among recombinant clones representing 120W. bancrofti parasites, prevalent across wide geographic areas of India and found to be highly conserved among them. Since it is highly conserved and plays an important role in Wolbachia cell division it appears to be a potential target for anti-filarial chemotherapy development.

Concepts: DNA, Protein, Amino acid, Diethylcarbamazine, Filariasis, Brugia timori, Brugia malayi, Wuchereria bancrofti

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Lymphatic filariasis is caused by three closely related nematode parasites: Wuchereria bancrofti, Brugia malayi and Brugia timori. These species have many ecological variants that differ in several aspects of their biology such as mosquito vector species, host range, periodicity, and morphology. Although the genome of B. malayi (the first genome sequenced from a parasitic nematode) has been available for more than five years, very little is known about genetic variability among the lymphatic dwelling filariae. The genetic diversity among these worms is not only interesting from a biological perspective, but it may have important practical implications for the Global Program to Eliminate Lymphatic Filariasis, as the parasites may respond differently to diagnostic tests and/or medical interventions. Therefore, better information on their genetic variability is urgently needed. With improved methods for nucleic acid extraction and recent advances in sequencing chemistry and instrumentation, this gap can be filled relatively inexpensively. Improved information on filarial genetic diversity may increase the chances of success for lymphatic filariasis elimination programs.

Concepts: Malaria, Nematodes, Vector, Diethylcarbamazine, Filariasis, Brugia timori, Brugia malayi, Wuchereria bancrofti

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SUMMARY Brugia malayi is one of the parasitic worms which causes lymphatic filariasis in humans. Its geographical distribution includes a large part of Asia. Despite its wide distribution, very little is known about the genetic variation and molecular epidemiology of this species. In this study, the internal transcribed spacer 1 (ITS1) nucleotide sequences of B. malayi from microfilaria-positive human blood samples in Northeast Borneo Island were determined, and compared with published ITS1 sequences of B. malayi isolated from cats and humans in Thailand. Multiple alignment analysis revealed that B. malayi ITS1 sequences from Northeast Borneo were more similar to each other than to those from Thailand. Phylogenetic trees inferred using Neighbour-Joining and Maximum Parsimony methods showed similar topology, with 2 distinct B. malayi clusters. The first cluster consisted of Northeast Borneo B. malayi isolates, whereas the second consisted of the Thailand isolates. The findings of this study suggest that B. malayi in Borneo Island has diverged significantly from those of mainland Asia, and this has implications for the diagnosis of B. malayi infection across the region using ITS1-based molecular techniques.

Concepts: DNA, Species, Sequence, Phylogenetic tree, Phylogenetics, Diethylcarbamazine, Brugia timori, Brugia malayi

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In a placebo controlled field trial, the effects of doxycycline (200mg/day) for 23 days followed by doxycycline (200mg/day) in combination with albendazole (ABZ) (400mg/day) for 7 days on depletion of Wolbachia endobacteria from Wuchereria bancrofti and microfilaricidal activity were studied in 68 patients (34 males and 34 females) from West Bengal, India. The drugs in combination (i.e., doxycycline+ABZ) provided the best efficacy by totally eliminating the circulating microfilaria (mf) (in 42% cases) on day 365 with (99.8%, P<0.05) suppression even on day 365 post-treatment compared to both exclusive doxycycline (69%, P<0.05) and ABZ (89%, P<0.05) groups. Thus, our results have established that a 30-day course of doxycycline in combination with a 7-day course of ABZ is sufficient to ensure long-term reduction in mf level by depleting Wolbachia from worm tissues. Doxycycline combined with ABZ led to a greater reduction in mf density in blood at 4 months (post-treatment) in comparison to doxycycline or ABZ alone. There were significant differences between the three treatments after 12 months (post-treatment). Further, the impact of a 7-day regimen of ABZ was surprisingly good in reducing mf compared to doxycycline-alone group. Adverse reactions were mild. A 30-day course of doxycycline and ABZ in combination is a safe and well-tolerated treatment for lymphatic filariasis with significant activity against microfilaremia.

Concepts: Wolbachia, Diethylcarbamazine, Filariasis, Doxycycline, Neglected diseases, Brugia malayi, Wuchereria bancrofti, Elephantiasis

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Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18-24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks.

Concepts: Nematodes, Diethylcarbamazine, Filariasis, Neglected diseases, Brugia timori, Brugia malayi, Wuchereria bancrofti, Elephantiasis

13

Many techniques for studying functional genomics of important target sites of anthelmintics have been restricted to Caenorhabditis elegans because they have failed when applied to animal parasites. To overcome these limitations, we have focused our research on the human nematode parasite Brugia malayi, which causes elephantiasis. Here, we combine single-cell PCR, whole muscle cell patch clamp, motility phenotyping (Worminator), and dsRNA for RNAi for functional genomic studies that have revealed, in vivo, four different muscle nAChRs (M-, L-, P-, and N-). The cholinergic anthelmintics had different selectivities for these receptors. We show that motility and patch-clamp responses to levamisole and pyrantel, but not morantel or nicotine, require the unc-38 and/or unc-29 genes. Derquantel behaved as a competitive antagonist and distinguished M-nAChRs activated by morantel (Kb 13.9 nM), P-nAChRs activated by pyrantel (Kb 126 nM), and L-nAChRs activated by levamisole (Kb 0.96 µM) and bephenium. Derquantel was a noncompetitive antagonist of nicotine, revealing N-type nAChRs. The presence of four diverse nAChRs on muscle is perhaps surprising and not predicted from the C. elegans model. The diverse nAChRs represent distinguishable drug targets with different functions: Knockdown of unc-38+unc-29 (L- and/or P-receptors) inhibited motility but knockdown of acr-16+acr-26 (M- and/or N-receptors) did not.

Concepts: Genome, RNA, Genomics, Caenorhabditis elegans, Animal, Nematode, Model organism, Brugia malayi

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Filarial nematodes currently infect up to 54 million people worldwide, with millions more at risk for infection, representing the leading cause of disability in the developing world. Brugia malayi is one of the causative agents of lymphatic filariasis and remains the only human filarial parasite that can be maintained in small laboratory animals. Many filarial nematode species, including B. malayi, carry an obligate endosymbiont, the alpha-proteobacteria Wolbachia, which can be eliminated through antibiotic treatment. Elimination of the endosymbiont interferes with development, reproduction, and survival of the worms within the mamalian host, a clear indicator that the Wolbachia are crucial for survival of the parasite. Little is understood about the mechanism underlying this symbiosis.

Concepts: Bacteria, Nematode, Diethylcarbamazine, Filariasis, Doxycycline, Brugia timori, Brugia malayi, Parthenogenesis

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Once interruption of transmission of lymphatic filariasis is achieved, morbidity prevention and management becomes more important. A study in Brugia malayi filariasis from India has shown sub-clinical lymphatic pathology with potential reversibility. We studied a Wuchereria bancrofti infected population, the major contributor to LF globally.

Concepts: Diethylcarbamazine, Filariasis, Brugia timori, Brugia malayi, Wuchereria bancrofti, Insect-borne diseases, Elephantiasis, Onchocercidae

4

Endemicity of lymphatic filariasis (LF) in Cambodia was proven in 1956 when microfilariae were detected in mosquitos in the Kratié province. In 2001, an extensive study confirmed the presence of both Brugia malayi and Wuchereria bancrofti microfilariae. In 2003, the Ministry of Health established a national task force to develop policies and strategies for controlling and eliminating neglected tropical diseases (NTDs), with the goal of eliminating LF by 2015. This article summarizes the work accomplished to eliminate LF as a public health problem in Cambodia.

Concepts: Malaria, Diethylcarbamazine, Filariasis, Neglected diseases, Brugia timori, Brugia malayi, Wuchereria bancrofti, Elephantiasis

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The filarial nematode Brugia malayi is an etiological agent of Lymphatic Filariasis. The capability of B. malayi and other parasitic nematodes to modulate host biology is recognized but the mechanisms by which such manipulation occurs are obscure. An emerging paradigm is the release of parasite-derived extracellular vesicles (EV) containing bioactive proteins and small RNA species that allow secretion of parasite effector molecules and their potential trafficking to host tissues. We have previously described EV release from the infectious L3 stage B. malayi and here we profile vesicle release across all intra-mammalian life cycle stages (microfilariae, L3, L4, adult male and female worms). Nanoparticle Tracking Analysis was used to quantify and size EVs revealing discrete vesicle populations and indicating a secretory process that is conserved across the life cycle. Brugia EVs are internalized by murine macrophages with no preference for life stage suggesting a uniform mechanism for effector molecule trafficking. Further, the use of chemical uptake inhibitors suggests all life stage EVs are internalized by phagocytosis. Proteomic profiling of adult male and female EVs using nano-scale LC-MS/MS described quantitative and qualitative differences in the adult EV proteome, helping define the biogenesis of Brugia EVs and revealing sexual dimorphic characteristics in immunomodulatory cargo. Finally, ivermectin was found to rapidly inhibit EV release by all Brugia life stages. Further this drug effect was also observed in the related filarial nematode, the canine heartworm Dirofilaria immitis but not in an ivermectin-unresponsive field isolate of that parasite, highlighting a potential mechanism of action for this drug and suggesting new screening platforms for anti-filarial drug development.

Concepts: Nematode, Nematodes, Diethylcarbamazine, Filariasis, Ivermectin, Brugia timori, Brugia malayi, Dirofilaria immitis