Diethylcarbamazine elicits Ca2+ signals through TRP-2 channels that are potentiated by emodepside in Brugia malayi muscles.

Filarial nematode infections are a major health concern in several countries. Lymphatic filariasis is caused by Wuchereria bancrofti and Brugia spp. affecting over 120 million people. Heavy infections can lead to elephantiasis, which has serious effects on individuals' lives. Although current anthelmintics are effective at killing microfilariae in the bloodstream, they have little to no effect against adult parasites found in the lymphatic system. The anthelmintic diethylcarbamazine is one of the central pillars of lymphatic filariasis control. Recent studies have reported that diethylcarbamazine can open transient receptor potential (TRP) channels in the muscles of adult female Brugia malayi, leading to contraction and paralysis. Diethylcarbamazine has synergistic effects in combination with emodepside on Brugia, inhibiting motility: emodepside is an anthelmintic that has effects on filarial nematodes and is under trial for the treatment of river blindness. Here, we have studied the effects of diethylcarbamazine on single Brugia muscle cells by measuring the change in Ca2+ fluorescence in the muscle using Ca2+-imaging techniques. Diethylcarbamazine interacts with the transient receptor potential channel, C classification (TRPC) ortholog receptor TRP-2 to promote Ca2+ entry into the Brugia muscle cells, which can activate Slopoke (SLO-1) Ca2+-activated K+ channels, the putative target of emodepside. A combination of diethylcarbamazine and emodepside leads to a bigger Ca2+ signal than when either compound is applied alone. Our study shows that diethylcarbamazine targets TRP channels to promote Ca2+ entry that is increased by emodepside activation of SLO-1 K+ channels.

Anthelmintic resistance and homeostatic plasticity (Brugia malayi).

Homeostatic plasticity refers to the capacity of excitable cells to regulate their activity to make compensatory adjustments to long-lasting stimulation. It is found across the spectrum of vertebrate and invertebrate species and is driven by changes in cytosolic calcium; it has not been explored in parasitic nematodes when treated with therapeutic drugs. Here we have studied the adaptation of Brugia malayi to exposure to the anthelmintic, levamisole that activates muscle AChR ion-channels. We found three phases of the Brugia malayi motility responses as they adapted to levamisole: an initial spastic paralysis; a flaccid paralysis that follows; and finally, a recovery of motility with loss of sensitivity to levamisole at 4 h. Motility, calcium-imaging, patch-clamp and molecular experiments showed the muscle AChRs are dynamic with mechanisms that adjust their subtype composition and sensitivity to levamisole. This homeostatic plasticity allows the parasite to adapt resisting the anthelmintic.

Multipurpose peptides: The venoms of Amazonian stinging ants contain anthelmintic ponericins with diverse predatory and defensive activities.

In the face of increasing drug resistance, the development of new anthelmintics is critical for controlling nematodes that parasitise livestock. Although hymenopteran venom toxins have attracted attention for applications in agriculture and medicine, few studies have explored their potential as anthelmintics. Here we assessed hymenopteran venoms as a possible source of new anthelmintic compounds by screening a panel of ten hymenopteran venoms against Haemonchus contortus, a major pathogenic nematode of ruminants. Using bioassay-guided fractionation coupled with liquid chromatography-tandem mass spectrometry, we identified four novel anthelmintic peptides (ponericins) from the venom of the neotropical ant Neoponera commutata and the previously described ponericin M-PONTX-Na1b from Neoponera apicalis venom. These peptides inhibit H. contortus development with IC50 values of 2.8-5.6 µM. Circular dichroism spectropolarimetry indicated that the ponericins are unstructured in aqueous solution but adopt α-helical conformations in lipid mimetic environments. We show that the ponericins induce non-specific membrane perturbation, which confers broad-spectrum antimicrobial, insecticidal, cytotoxic, hemolytic, and algogenic activities, with activity across all assays typically correlated. We also show for the first time that ponericins induce spontaneous pain behaviour when injected in mice. We propose that the broad-spectrum activity of the ponericins enables them to play both a predatory and defensive role in neoponeran ants, consistent with their high abundance in venom. This study reveals a broader functionality for ponericins than previously assumed, and highlights both the opportunities and challenges in pursuing ant venom peptides as potential therapeutics.

The Eagle effect in the Wolbachia-worm symbiosis.

BACKGROUND: Onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) are two human neglected tropical diseases that cause major disabilities. Mass administration of drugs targeting the microfilarial stage has reduced transmission and eliminated these diseases in several countries but a macrofilaricidal drug that kills or sterilizes the adult worms is critically needed to eradicate the diseases. The causative agents of onchocerciasis and lymphatic filariasis are filarial worms that harbor the endosymbiotic bacterium Wolbachia. Because filarial worms depend on Wolbachia for reproduction and survival, drugs targeting Wolbachia hold great promise as a means to eliminate these diseases. METHODS: To better understand the relationship between Wolbachia and its worm host, adult Brugia pahangi were exposed to varying concentrations of doxycycline, minocycline, tetracycline and rifampicin in vitro and assessed for Wolbachia numbers and worm motility. Worm motility was monitored using the Worminator system, and Wolbachia titers were assessed by qPCR of the single copy gene wsp from Wolbachia and gst from Brugia to calculate IC50s and in time course experiments. Confocal microscopy was also used to quantify Wolbachia located at the distal tip region of worm ovaries to assess the effects of antibiotic treatment in this region of the worm where Wolbachia are transmitted vertically to the microfilarial stage. RESULTS: Worms treated with higher concentrations of antibiotics had higher Wolbachia titers, i.e. as antibiotic concentrations increased there was a corresponding increase in Wolbachia titers. As the concentration of antibiotic increased, worms stopped moving and never recovered despite maintaining Wolbachia titers comparable to controls. Thus, worms were rendered moribund by the higher concentrations of antibiotics but Wolbachia persisted suggesting that these antibiotics may act directly on the worms at high concentration. Surprisingly, in contrast to these results, antibiotics given at low concentrations reduced Wolbachia titers. CONCLUSION: Wolbachia in B. pahangi display a counterintuitive dose response known as the "Eagle effect." This effect in Wolbachia suggests a common underlying mechanism that allows diverse bacterial and fungal species to persist despite exposure to high concentrations of antimicrobial compounds. To our knowledge this is the first report of this phenomenon occurring in an intracellular endosymbiont, Wolbachia, in its filarial host.

A novel assay to isolate and quantify third-stage Dirofilaria immitis and Brugia malayi larvae emerging from individual Aedes aegypti.

BACKGROUND: Mosquitoes transmit filarial nematodes to both human and animal hosts, with worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood-feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector. METHODS: We developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 °C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi-infected Ae. aegypti. RESULTS: Approximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi produced emerging third-stage larvae. Once D. immitis third-stage larvae emerged at 13 days post infection, the proportion of mosquitoes producing them and the number produced per mosquito remained stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12-14 post infection. Increased uptake of D. immitis microfilariae increased the fitness cost to the mosquito but did not increase the number of emerging third-stage larvae. CONCLUSIONS: We provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious third-stage larvae for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interactions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission.

Ivermectin inhibits extracellular vesicle secretion from parasitic nematodes.

Lymphatic filariasis (LF) is a disease caused by parasitic filarial nematodes that is endemic in 49 countries of the world and affects or threatens over 890 million people. Strategies to control LF rely heavily on mass administration of anthelmintic drugs including ivermectin (IVM), a macrocyclic lactone drug considered an Essential Medicine by the WHO. However, despite its widespread use the therapeutic mode of action of IVM against filarial nematodes is not clear. We have previously reported that filarial nematodes secrete extracellular vesicles (EVs) and that their cargo has immunomodulatory properties. Here we investigate the effects of IVM and other anti-filarial drugs on parasitic nematode EV secretion, motility, and protein secretion. We show that inhibition of EV secretion was a specific property of IVM, which had consistent and significant inhibitory effects across nematode life stages and species, with the exception of male parasites. IVM inhibited EV secretion, but not parasite motility, at therapeutically relevant concentrations. Protein secretion was inhibited by IVM in the microfilariae stage, but not in any other stage tested. Our data provides evidence that inhibiting the secretion of immunomodulatory EVs by parasitic nematodes could explain, at least in part, IVM mode of action and provides a phenotype for novel drug discovery.

Prediction pipeline for discovery of regulatory motifs associated with Brugia malayi molting.

Filarial nematodes can cause debilitating diseases in humans. They have complicated life cycles involving an insect vector and mammalian hosts, and they go through a number of developmental molts. While whole genome sequences of parasitic worms are now available, very little is known about transcription factor (TF) binding sites and their cognate transcription factors that play a role in regulating development. To address this gap, we developed a novel motif prediction pipeline, Emotif Alpha, that integrates ten different motif discovery algorithms, multiple statistical tests, and a comparative analysis of conserved elements between the filarial worms Brugia malayi and Onchocerca volvulus, and the free-living nematode Caenorhabditis elegans. We identified stage-specific TF binding motifs in B. malayi, with a particular focus on those potentially involved in the L3-L4 molt, a stage important for the establishment of infection in the mammalian host. Using an in vitro molting system, we tested and validated three of these motifs demonstrating the accuracy of the motif prediction pipeline.

Emodepside has sex-dependent immobilizing effects on adult Brugia malayi due to a differentially spliced binding pocket in the RCK1 region of the SLO-1 K channel.

Filariae are parasitic nematodes that are transmitted to their definitive host as third-stage larvae by arthropod vectors like mosquitoes. Filariae cause diseases including: lymphatic filariasis with distressing and disturbing symptoms like elephantiasis; and river blindness. Filarial diseases affect millions of people in 73 countries throughout the topics and sub-tropics. The drugs available for mass drug administration, (ivermectin, albendazole and diethylcarbamazine), are ineffective against adult filariae (macrofilariae) at the registered dosing regimen; this generates a real and urgent need to identify effective macrofilaricides. Emodepside, a veterinary anthelmintic registered for treatment of nematode infections in cats and dogs, is reported to have macrofilaricidal effects. Here, we explore the mode of action of emodepside using adult Brugia malayi, one of the species that causes lymphatic filariasis. Whole-parasite motility measurement with Worminator and patch-clamp of single muscle cells show that emodepside potently inhibits motility by activating voltage-gated potassium channels and that the male is more sensitive than the female. RNAi knock down suggests that emodepside targets SLO-1 K channels. We expressed slo-1 isoforms, with alternatively spliced exons at the RCK1 (Regulator of Conductance of Potassium) domain, heterologously in Xenopus laevis oocytes. We discovered that the slo-1f isoform, found in muscles of males, is more sensitive to emodepside than the slo-1a isoform found in muscles of females; and selective RNAi of the slo-1a isoform in female worms increased emodepside potency. In Onchocerca volvulus, that causes river blindness, we found two isoforms in adult females with homology to Bma-SLO-1A and Bma-SLO-1F at the RCK1 domain. In silico modeling identified an emodepside binding pocket in the same RCK1 region of different species of filaria that is affected by these splice variations. Our observations show that emodepside has potent macrofilaricidal effects and alternative splicing in the RCK1 binding pocket affects potency. Therefore, the evaluation of potential sex-dependent effects of an anthelmintic compound is of importance to prevent any under-dosing of one or the other gender of nematodes once given to patients.

Recognition and killing of Brugia malayi microfilariae by human immune cells is dependent on the parasite sample and is not altered by ivermectin treatment.

Mass administration of macrocyclic lactones targets the transmission of the causative agents of lymphatic filariasis to their insect vectors by rapidly clearing microfilariae (Mf) from the circulation. It has been proposed that the anti-filarial action of these drugs may be mediated through the host immune system. We recently developed an in vitro assay for monitoring the attachment to and killing of B. malayi Mf by human neutrophils (PMNs) and monocytes (PBMCs), however, the levels of both cell to worm attachment and leukocyte mediated Mf killing varied greatly between individual experiments. To determine whether differences in an individual's immune cells or the Mf themselves might account for the variability in survival, PMNs and PBMCs were isolated from 12 donors every week for 4 weeks and the cells used for survival assays with a different batch of Mf, thereby keeping donors constant but varying the Mf sample. Results from these experiments indicate that, overall, killing is Mf-rather than donor-dependent. To assess whether ivermectin (IVM) or diethylcarbamazine (DEC) increase killing, Mf were incubated either alone or with immune cells in the presence of IVM or DEC. Neither drug induced a significant difference in the survival of Mf whether cultured with or without cells, with the exception of DEC at 2 h post incubation. In addition, human PBMCs and PMNs were incubated with IVM or DEC for 1 h or 16 h prior to RNA extraction and Illumina sequencing. Although donor-to-donor variation may mask subtle differences in gene expression, principle component analysis of the RNASeq data indicates that there is no significant change in the expression of any genes from the treated cells versus controls. Together these data suggest that IVM and DEC have little direct effect on immune cells involved in the rapid clearance of Mf from the circulation.

Immune responses of Aedes togoi, Anopheles paraliae and Anopheles lesteri against nocturnally subperiodic Brugia malayi microfilariae during migration from the midgut to the site of development.

BACKGROUND: Lymphatic filariasis is a mosquito-borne disease caused by filarioid nematodes. A comparative understanding of parasite biology and host-parasite interactions can provide information necessary for developing intervention programmes for vector control. Here, to understand such interactions, we choose highly susceptible filariasis vectors (Aedes togoi and Anopheles lesteri) as well as Anopheles paraliae, which has lower susceptibility, infected them with nocturnally subperiodic (NSP) Brugia malayi microfilariae (mf) and studied the exsheathment, migration and innate immune responses among them. METHODS: Mosquito-parasite relationships were systematically investigated from the time mf entered the midgut until they reached their development site in the thoracic musculature (12 time points). RESULTS: Results showed that exsheathment of B. malayi mf occurred in the midgut of all mosquito species and was completed within 24 h post-blood meal. The migration of B. malayi mf from the midgut to thoracic muscles of the highly susceptible mosquitoes Ae. togoi and An. lesteri was more rapid than in the low susceptibility mosquito, An. paraliae. Melanisation and degeneration, two distinct refractory phenotypes, of mf were found in the midgut, haemocoel and thoracic musculature of all mosquito species. Melanisation is a complex biochemical cascade that results in deposition of melanin pigment on a capsule around the worms. Also, some biological environments in the body are inhospitable to parasite development and cause direct toxicity that results in vacuolated or degenerated worms. Even though Ae. togoi is highly susceptible to B. malayi, melanisation responses against B. malayi mf were first noted in the haemocoel of Ae. togoi, followed by a degeneration process. In contrast, in An. lesteri and An. paraliae, the degeneration process occurred in the haemocoel and thoracic musculature prior to melanisation responses. CONCLUSION: This study provides a thorough description of the comparative pathobiology of responses of mosquitoes against the filarial worm B. malayi.

Similarities and differences between helminth parasites and cancer cell lines in shaping human monocytes: Insights into parallel mechanisms of immune evasion.

A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma) or to live microfilariae (mf) of Brugia malayi-a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1ß), M2-associated (CCL13, CD206), Mreg-associated (IL-10, TGF-ß), and angiogenesis associated (MMP9, VEGF) genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1ß, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.

Profiling extracellular vesicle release by the filarial nematode Brugia malayi reveals sex-specific differences in cargo and a sensitivity to ivermectin.

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.

Crystal structure of Brugia malayi venom allergen-like protein-1 (BmVAL-1), a vaccine candidate for lymphatic filariasis.

Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1 Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitate-binding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules.

Brugia malayi microfilariae adhere to human vascular endothelial cells in a C3-dependent manner.

Brugia malayi causes the human tropical disease, lymphatic filariasis. Microfilariae (Mf) of this nematode live in the bloodstream and are ingested by a feeding mosquito vector. Interestingly, in a remarkable co-evolutionary adaptation, Mf appearance in the peripheral blood follows a circadian periodicity and reaches a peak when the mosquito is most likely to feed. For the remaining hours, the majority of Mf sequester in the lung capillaries. This circadian phenomenon has been widely reported and is likely to maximise parasite fitness and optimise transmission potential. However, the mechanism of Mf sequestration in the lungs remains largely unresolved. In this study, we demonstrate that B. malayi Mf can, directly adhere to vascular endothelial cells under static conditions and under flow conditions, they can bind at high (but not low) flow rates. High flow rates are more likely to be experienced diurnally. Furthermore, a non-periodic nematode adheres less efficiently to endothelial cells. Strikingly C3, the central component of complement, plays a crucial role in the adherence interaction. These novel results show that microfilariae have the ability to bind to endothelial cells, which may explain their sequestration in the lungs, and this binding is increased in the presence of inflammatory mediators.

Immunoprophylaxis of multi-antigen peptide (MAP) vaccine for human lymphatic filariasis.

Human lymphatic filariasis, the parasitic disease caused by the filarial nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori, is ranked as the second most complex clinical condition leading to permanent and long-term disability. The multiple antigen peptide (MAP) approach is an effective method to chemically synthesize and deliver multiple T and B cell epitopes as the constituents of a single immunogen. Here, we report on the design, chemical synthesis, and immunoprophylaxis of three epitopes that have been identified from promising vaccine candidates reported in our previous studies, constructed as MAP on an inert lysine core for human lymphatic filariasis in Jird model. Two epitopes from Thioredoxin and one epitope from Transglutaminase were constructed as MAP in an inert lysine core. The immunoprophylaxis of the synthetic vaccine construct studied in Jird models showed protective antibody (1 in 64,000 titer) and cellular immune response. Thioredoxin-Transglutaminase MAP (TT MAP) conferred a significantly high protection of 63.04% compared to control (8.5%). Multi-antigen peptide vaccine is one best approach to provide immunity against multiple antigens delivered by the complex filarial parasite.

Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis.

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.

Brugia malayi microfilariae transport alphaviruses across the mosquito midgut.

Concurrent ingestion of microfilariae (MF) and arboviruses by mosquitoes can enhance mosquito transmission of virus compared to when virus is ingested alone. Within hours of being ingested, MF penetrate the mosquito midgut and introduce virus into mosquito hemocoel, creating a disseminated viral infection much sooner than normal. How virus is actually introduced is not known. In this report, we present experimental evidence that suggests that certain alphaviruses may adhere or otherwise associate with sheathed Brugia malayi MF in the blood of a dually-infected host and that the virus is carried into the mosquito hemocoel by the MF during their penetration of the mosquito midgut. The mechanism of MF enhancement may be more complex than simple leakage of viremic blood into the hemocoel during MF penetration. The affinity of arboviruses to adhere to or otherwise associate with MF may depend on the specific combination of the virus and MF involved in a dual host infection. This in turn may determine the relative importance that MF enhancement has within an arbovirus transmission system.

Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release.

BACKGROUND: Wuchereria bancrofti, Brugia malayi and Brugia timori infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases. PRINCIPAL FINDINGS: In vitro, PMNs expel large amounts of NETs that capture but do not kill B. malayi Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill B. malayi Mf in vitro and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf in vitro, which may reflect the pathological heterogeneity associated with filarial parasitic infections. CONCLUSIONS/SIGNIFICANCE: Human innate immune cells are able to recognize, attach to and kill B. malayi microfilariae in an in vitro system. This suggests that, in vivo, the parasites can evade this ability, or that only some human hosts support an infection with circulating Mf.

Filarial infection influences mosquito behaviour and fecundity.

Understanding vector-parasite interactions is increasingly important as we move towards the endpoint goals set by the Global Programme for the Elimination of Lymphatic Filariasis (GPELF), as interaction dynamics may change with reduced transmission pressure. Elimination models used to predict programmatic endpoints include parameters for vector-specific transmission dynamics, despite the fact that our knowledge of the host-seeking behaviour of filariasis infected mosquitoes is lacking. We observed a dynamic, stage-specific and density dependent change in Aedes aegypti behaviour towards host cues when exposed to Brugia malayi filarial parasites. Infected mosquitoes exhibited reduced activation and flight towards a host during the period of larval development (L1/L2), transitioning to a 5 fold increase in activation and flight towards a host when infective stage larvae (L3) were present (p < 0.001). In uninfected control mosquitoes, we observed a reduction in convergence towards a host during the same period. Furthermore, this behaviour was density dependent with non-activated mosquitoes harbouring a greater burden of L1 and L2 larvae while activated mosquitoes harboured a greater number of L3 (p < 0.001). Reductions in fecundity were also density-dependent, and extended to mosquitoes that were exposed to microfilariae but did not support larval development.

Can community-based integrated vector control hasten the process of LF elimination?

Community-based integrated vector control (IVC) using polystyrene beads (EPS) and pyrethroid impregnated curtains (PIC) as an adjunct to mass drug administration (MDA) was implemented for lymphatic filariasis elimination, in the filaria endemic villages of Tirukoilur, south India. In all the villages, MDA was carried out by the state health machinery, as part of the national filariasis elimination programme. Thirty-six difficult-to-control villages were grouped as, viz, MDA alone, MDA + EPS and MDA + EPS + PIC arms. Implementation and monitoring of IVC was carried out by the community. After 3 years of IVC, higher reductions in filariometric indices were observed in both the community and vector population. Decline in antigenaemia prevalence was higher in MDA + IVC as compared to MDA alone arm. Vector density dropped significantly (P < 0.05) in both the IVC arms, and nil transmission was observed during post-IVC period. Almost 53.8 and 75.8 % of the cesspits in MDA + EPS and MDA + EPS + PIC arms were closed by the householders, due to the enhanced awareness on vector breeding. The paper presents the key elements of IVC implementation through social mobilization in a LF prevalent area. Thus, community-based IVC strategy can hasten LF elimination, as it reduced the transmission and filariometric indices significantly. Indices were maintained at low level with nil transmission, by the community through IVC tools.