RNAi-mediated silencing of ARV1 in Setaria digitata impairs in-vitro microfilariae release, embryogenesis and adult parasite viability.

Setaria digitata is a nematode that resides in the peritoneal cavity of ruminants causing cerebrospinal nematodiasis disease affecting livestock and inflicting significant economic forfeitures in Asia. Further, this nematode can infect humans, causing abscesses, allergic reactions, enlarged lymph nodes, eye lesions and inflammation of the lungs. The 'ARE2 required for viability1' (ARV1) encodes for putative lipid transporter localized in the endoplasmic reticulum (ER) and Golgi complex membrane in humans and yeast. In the present study, the functional role of S. digitata ARV1 (SD-ARV1) was investigated using RNA interference (RNAi) reverse genetic tool. The targeted silencing SD-ARV1 transcripts by siRNA mediated RNAi resulted in a dramatic reduction of SD-ARV1 gene and protein expressions in S. digitata, which in turn modulated the parasitic motility, its production of eggs and microfilaria viability. Further, the same silencing caused severe phenotypic deformities such as distortion of eggs and embryonic development arrest in the intrauterine stages of adult female S. digitata. These results suggest that SD-ARV1 plays a pivotal role in worm embryogenesis, adult parasite motility and microfilariae viability. Finally, the ubiquitous presence of ARV1 in human filarial nematodes, its crucial functional roles in nematode biology and its remarkable diversity in primary protein structure compared to homologues in their hosts warrants further investigations to ascertain its candidacy in anthelmintic drug development.

Influence of autophagy, apoptosis and their interplay in filaricidal activity of C-cinnamoyl glycosides.

The present work aims to explore the mechanism of action of C-cinnamoyl glycoside as an antifilarial agent against the bovine filarial nematode Setaria cervi. Both apoptosis and autophagy programmed cell death pathways play a significant role in parasitic death. The generation of reactive oxygen species, alteration of the level of antioxidant components and disruption of mitochondrial membrane potential may be the causative factors that drive the parasitic death. Monitoring of autophagic flux via the formation of autophagosome and autophagolysosome was detected via CYTO ID dye. The expression profiling of both apoptotic and autophagic marker proteins strongly support the initial findings of these two cell death processes. The increased interaction of pro-autophagic protein Beclin1 with BCL-2 may promote apoptotic pathway by suppressing anti-apoptotic protein BCL-2 from its function. This in turn partially restrains the autophagic pathway by engaging Beclin1 in the complex. But overall positive increment in autophagic flux was observed. Dynamic interaction and regulative balance of these two critical cellular pathways play a decisive role in controlling disease pathogenesis. Therefore, the present experimental work may prosper the chance for C-cinnamoyl glycosides to become a potential antifilarial therapeutic in the upcoming day after detail in vivo study and proper clinical trial.

Functional analysis of a novel parasitic nematode-specific protein of Setaria digitata larvae in Culex quinquefasciatus by siRNA mediated RNA interference.

BACKGROUND: Functional analysis of animal parasitic nematode genes is often quite challenging due to the unavailability of standardised in vitro culture conditions and lack of adequate tools to manipulate these genes. Therefore, this study was undertaken to investigate the suitability of Culex quinquefasciatus, as an in vivo culture platform for Setaria digitata larvae and RNA interference (RNAi), as a post-transcriptional gene silencing tool to study the roles of a vital gene that encodes a novel parasitic nematode-specific protein (SDNP). RESULTS: The red colour fluorescence detected following RNAi injection to the thorax of C. quinquefasciatus indicated the uptake of dsRNA by S. digitata larvae. The reduction of SDNP transcripts in siRNA treated larvae compared to non-treated larvae, as determined by qPCR, indicated that the siRNA pathway is operational in S. digitata larvae. The observation of motility reductions and deformities during the development indicated the association of SDNP in larvae locomotion and development processes, respectively. The irregularities in the migration of larvae in mosquitoes and elevated survival rates of mosquitoes compared to their untreated counterparts indicated reduced parasitism of S. digitata larvae in mosquitoes upon targeted downregulation of SDNP by siRNA treatment. CONCLUSION: SDNP plays vital roles in muscle contraction, locomotion, development processes, larval development and parasitism of S. digitata. Its ubiquitous presence in parasitic nematodes and its absence in their hosts provide a tantalising prospect of the possibility of targeting SDNP for future development of anthelmintic drugs. The susceptibility of the larval stages of S. digitata for RNAi in Culex quinquefasciatus was also demonstrated for the first time in this study.

Filarial nematode (order: Spirurida) surveillance in urban habitats, in the city of Pécs (Hungary).

As part of the seasonal mosquito control activities in the city of Pécs (Baranya County, Hungary), a total of 1123 adult female mosquitoes belonging to 18 species (including the invasive species Aedes koreicus) were collected from human-inhabited areas, using CO2-baited traps, during two consecutive years. To survey the presence and prevalence of filarial parasites in these mosquitoes, we performed a molecular survey for filarial DNA, attempted by PCR using generic primers (COI), and followed by DNA sequencing. Filaroid nematode DNA was detected in 4% of investigated mosquito pools. Out of 410 pools, 9 pools of mosquitoes were positive for Dirofilaria repens (Aedes vexans, Aedes koreicus, Coquillettidia richiardii), and/or Dirofilaria immitis (Ae. vexans, Cq. richiardii), and further 8 pools were positive for Setaria tundra (Ae. vexans, Cq. richiardii). Our study provides novel insight for prevalence of filaroid nematodes in mosquitoes occurring in close proximity to humans, thereby highlights the possible human and veterinary health importance of these mosquito species, including the recently introduced invasive mosquito Ae. koreicus.

Development of siRNA mediated RNA interference and functional analysis of novel parasitic nematode-specific protein of Setaria digitata.

Despite the differences of the host, parasitic nematodes may share commonalities in their parasitizing genes. Setaria digitata novel protein (SDNP) is such an entity which is parasitic nematode-specific and having sequence similarities with those of W. bancrofti, B. malayi, Loa loa and Onchocerca volvulus. Post-transcriptional gene silencing by siRNA mediated RNA interference (RNAi) is a widely used technique in functional genomics. Though the technique has been used in several free-living, plant and animal parasitic nematodes, it has not yet been tried out for the filarial worm S. digitata. In this study, we developed an effective siRNA delivery method by microinjection and utilized the siRNAi tool to knockdown SDNP to study the phenotypic and cellular changes associated with the interference. qPCR analysis revealed, a significant reduction of SDNP transcript levels following siRNA microinjection into S. digitata adult worms. Similarly, immunohistochemical staining indicated a reduction of SDNP protein expression. Furthermore, worms treated with siRNA showed a significant reduction of microfilariae release together with embryonic lethality by arresting an early developmental stage compared to non-treated worms. A distinct motility reduction was also observed in treated worms compared to non-treated counterparts. This is the first report of the amenability of S. digitata to the siRNA induced RNAi. The presence of inter-domain linkers of muscle-specific twitchin kinase and calcium-dependent protein kinase isoform CDPK1 together with what our results revealed suggest that SDNP is most likely a protein involved in muscle movement and growth and development of the nematode. Hence SDNP has the characteristics of a potential drug target.

Exploration of antifilarial activity of gold nanoparticle against human and bovine filarial parasites: A nanomedicinal mechanistic approach.

The present work seeks to explore the antifilarial activity of biopolymer functionalized gold nanoparticles (AuNPs) against human filarial parasite (Wuchereria bancrofti) through Nrf2 signaling for the first time. A natural polymer, chitosan is used along with Terminalia chebula extract to synthesize AuNPs following the principles of green chemistry. The probable mode of action of AuNPs as filaricidal agent has been investigated in detail using model filarial parasite, Setaria cervi (bovine parasite). Biopolymers inspired AuNPs exhibit superior antifilarial activity against both human and bovine filarial parasites, and are able to induce oxidative stress and apoptotic cell death in filarial parasites mediated through mitochondria. AuNPs also alter the Nrf2 signaling. In addition, the synthesized nanomaterials appear to be nontoxic to mammalian system. Thus the present mechanistic study, targeting human filarial parasites, has the potential to increase the therapeutic prospects of AuNPs to control lymphatic filariasis in the upcoming days.

Presence of ecto-protein tyrosine phosphatase activity is vital for survival of Setaria cervi, a bovine filarial parasite.

The ecto protein tyrosine phosphatases (PTP) are known to play a crucial role in the pathogenesis and survival of the intracellular parasites. However, their presence and role in filarial parasites is still unknown. We found a significant amount of tyrosine phosphatase activity in the surface antigen fraction extracted from Setaria cervi (S. cervi), a bovine filarial parasite. An antibody designed against the conserved catalytic core of human protein tyrosine phosphatases, PTP1B cross reacted with a 63 kDa band in the surface antigen. We detected a significant amount of PTP activity in the intact S. cervi adult parasites as well as microfilariae in this study for the first time. This PTP may be localized on the surface of the parasite with an exposed active site available for the external substrates. The PTP activity was also inhibited by sodium orthovanadate and phenyl arsine oxide, specific inhibitors of PTP in both the life stages. The Km and Vmax for PTP in the adult parasites and microfilariae were determined to be 2.574 ± 0.14 mM; 206.3 ± 2.75 µM Pi/h/two parasites and 5.510 ± 0.59 mM; 62.27 ± 2.27 µM Pi/h/10(6) parasites respectively using O-P-L-Tyrosine as substrate. Interestingly, a positive correlation was observed between the inhibition in PTP activity and reduction in the motility/ viability of the parasites when they were subjected to the specific PTP inhibitors (Orthovanadate and Phenyl arsine oxide) for 4 h in the KRB maintenance medium. The activity was also significantly inhibited in the parasites exposed to antifilarial drug/compounds for e.g. Diethylcarbamazine, Acetylsalicylic Acid and SK7, a methyl chalcone. Therefore suggesting a possible role played by PTP in the survival of the parasite, its interaction with the host as well as in the screening of newly synthesized antifilarials/drugs.

Antifilarial activity of ethyl acetate extract of Vitex negundo leaves in vitro.

OBJECTIVE: To evaluate the possible antifilarial effect of ethyl acetate extract of Vitex negundo (Verbenaceae) leaves against Setaria cervi filarial parasite in vitro. METHODS: In vitro screening was done by the method of motility inhibition and MTT reduction assay with concentrations of 0.03 to 1.00 mg/mL for 2 to 24 h incubation periods respectively, for possible antifilarial effect by comparing with control. RESULTS: In motility assay, complete inhibition of motility was observed and in MTT reduction assay which gave >50% reduction for concentrations 0.20, 0.50 and 1.00 mg/mL at 10, 6 and 2 h incubation periods respectively in a dose dependent manner (P<0.05). An antifilarial effect imparted by plant extract was found to be a function of their relative concentrations. Inhibitory concentration (IC50) for the plant extract was found to be 0.16 mg/mL. CONCLUSIONS: The present study recorded significant antifilarial effect of Vitex negundo plant extract and contributed toward the development of database for novel drug candidates for lymphatic filariasis.

Biochemical composition and metabolic pathways of filarial worms Setaria cervi: search for new antifilarial agents.

The main problem regarding the chemotherapy of filariasis is that no safe and effective drug is available yet to combat the adult human filarial worms. Setaria cervi, the causal organism of setariasis and lumbar paralysis in cattle, is routinely employed as a model organism for conducting biochemical and enzymatic studies on filarial parasites. In view of the practical difficulties in procuring human strains of Wuchereria bancrofti and Brugia malayi for drug screening, the bovine filarial parasite S. cervi, resembling the human species in having microfilarial periodicity and chemotherapeutic response to known antifilarial agents, is widely used as a model in such studies. For a rational approach to antifilarial chemotherapy, knowledge of the biochemical composition and metabolic pathways of this helminth parasite may be of paramount importance, so that more potent antifilarial agents based on specific drug targets can be identified in drug discovery programmes. The present review provides an update on the biochemistry of the important metabolic pathways functioning within this potentially important bovine parasite, that have so far been studied, and on those that need to be investigated further so as to identify novel drug targets that can be exploited for designing new antifilarial drugs.

The filarial endosymbiont Wolbachia sp. is absent from Setaria equina.

Wolbachia sp. was first reported in filarial nematodes over 25 yr ago. Today, much research is focused on the role of these bacteria in filarial worm biology. The filarial symbionts are closely related to arthropod symbionts, which are known to modify host reproduction and biology through various mechanisms. Similarly, it has been suggested that Wolbachia sp. is essential for long-term survival and reproduction of filariae. We report that Wolbachia sp. 16S rDNA was not found in the equine filarial nematode Setaria equina, using either polymerase chain reaction (PCR) or DNA hybridization. In addition, ultrastructural analysis of adult worms did not reveal the presence of Wolbachia sp. in hypodermal cords or reproductive tissues. These data suggest that like Onchocerca flexuosa and Acanthocheilonema vitae, S. equina may not be dependent on Wolbachia sp. for survival.

Voltage activated calcium channels in somatic muscle of filarial nematode Setaria cervi.

Acetylcholine (Ach), levamisole and pyrantel pamoate all cause stimulation of spontaneous rhythmic movements of whole worm and nerve muscle preparation of filarial nematode Setaria cervi. These stimulant effects are manifested only in the presence of available Ca2+ or extracellular Ca2+. Electrical stimulation of nerve muscle preparation of Setaria cervi elicited depolarization and increase in amplitude and tone of contractions. Electrical current stimulates Ca2+ entry leading to depolarization and during the phase of depolarization addition of any of the three stimulants viz. Ach, levamisole or pyrantel pamoate fails to elicit any response on nerve muscle preparation. The findings indicate that electrical stimulation, excitatory neurotransmitter Ach and stimulant anthelmintics levamisole and pyrantel pamoate all produce their stimulant effect by triggering entry of Ca2+ into the muscle cell. Further, blocking the calcium channels by nifedepine and thereby the entry of Ca2+ into the cells blocks the stimulant effect of Ach levamisole and pyrantel pamoate.

Quinone analogue irrecoverably paralyses the filarial parasites in vitro.

2,3-Dimethoxy-5-methyl-1,4-benzoquinone (Q0), an analogue of ubiquinone, irreversibly paralyses the adult and microfilariae of the cattle filarial parasite Setaria digitata. The same concentration of Q0 that paralyses the microfilariae of S. digitata also paralyses the microfilariae of the human filarial parasite Wuchereria bancrofti within the same duration. Thus the experiments done in the model S. digitata system can well be extended to the human filarial system. A drug at the level of the quinone-centered energy generating system, perhaps an analogue of quinone like Q0, can inactivate the filarial parasites and may prove to be an effective drug to control filariasis.

Antifilarial activity of Mallotus philippensis Lam. on Setaria cervie (Nematoda: Filarioidea) in vitro.

The effect of aqueous and alcoholic extracts of the leaves of Mallotus philippensis (Lam.) Muell. Arg. was studied on the spontaneous movements of the whole worm and nerve-muscle (n.m.) preparation of Setaria cervi and on the survival of microfilariae in vitro. Both the extracts caused inhibition of spontaneous motility of whole worm and the n.m. preparation of S. Cervi characterized by initial stimulation followed by depression in amplitude. The tone and rate of contractions remained visibly unaffected. Aqueous extract at higher concentration showed immediate reduction in tone. The concentration required to inhibit the movements of n.m. preparation was 1/5th for aqueous and 1/11th for alcoholic extract compared to that for the whole worm, suggesting a cuticular permeability barrier. The stimulatory response of acetylcholine was blocked by aqueous extract on whole worm movements. On the microfilariae the LC50 and LC90 were 18 and 20 ng/ml for aqueous and 12 and 15 ng/ml for alcoholic extracts respectively.

A consideration on the relationship between worm age and mortality of Setaria marshalli recovered from the peritoneal cavity of calves born in Tottori prefecture, Japan.

Eight calves born in November-December of 1992 were necropsied at the age of 29-230 days in an attempt to estimate the life span of Setaria marshalli in Japan. Worm ages were estimated on the base of active season for mosquitoes. Thirty worms estimated 4-9 months old recovered from 4 calves were all alive in the peritoneal cavity. Thirty-five out of 50 worms estimated 7-13 months old were dead with being entrapped by fibrin on the peritoneum. It is speculated that this is a normal fate of this parasite at the end of its life, and therefore, the life span of S. marshalli would be approximately one year after prenatal infection.

Histological changes in the spleen of BALB/C mice caused by excretory-secretory protein(s) of Setaria digitata (Von Linstow).

The hatching associated materials generally called the excretory-secretory material (ES) released from the filarial parasite S. digitata along with the microfilariae (mf) under in vitro conditions showed immunosuppression. However the protein from lysate of embryo zone, from which ES materials were released along with the mf showed immunopotentiation in BALB/C mice. The histological analysis of spleen of immunized BALB/C mice showed changes confirming the in vitro findings. The main changes observed in the suppressed spleen were decrease in size and number of lymph follicles and increase in number of reticuloendothelial (RE) cells and cosinophil cells, whereas the main change in the potentiated spleen was an increase in the number of lymph follicles as compared to control spleen. These results clearly indicate that there are two types of ES materials in S. digitata, the native material present in the mature embryo causes immunopotentiation, and the materials released during the hatching process into the hostile environment along with mf causes immunosuppression. The difference in the biological effect between the materials released by lysis and hatching of the embryo opens up fresh understanding about the survival strategies of filarial parasites.

Identification of voltage-sensitive calcium channels in Setaria cervi (Nematoda: Filarioidea) and their role in regulation of spontaneous motility.

Whole worm of Setaria cervi and its nerve muscle preparation exhibit rhythmical movements when suspended in modified Ringer's solution in an isolated organ bath. Deprivation of calcium from the bathing fluid results in gradual reduction in the amplitude and rate of contraction till the movements ceased completely. Similar results were obtained by adding EDTA (5 micrograms/ml) to the bath. The effect was concentration related and was evident early with higher concentration of EDTA. Acetylcholine which increases spontaneous movements of the whole worm as well as N.M. preparation failed to do so when the movements were inhibited either in calcium free solution or in the presence of EDTA. Addition of calcium channel blocker, Nifedipine to the bathing fluid also resulted in concentration related reduction of movements of the preparation of Setaria. Further, the stimulant response of Ach was also reduced by Nifedipine and this too was related to the concentration of calcium channel blocker in the bath. The evidence clearly indicates that presence of calcium is essential for the stimulant response of Ach on S. cervi, which like mammalian tissues contain calcium channels that can be blocked by specific blocking agents.

Interaction of filarial proteins on growth regulation of normal lung epithelial cells in vitro.

An in vitro model to examine the effects of filarial proteins on lung epithelial cells has been developed. Several of these proteins appear in circulation of infected individuals. A close association between tropical pulmonary eosinophilia (TPE) and filariasis has been reported by several workers. [3H]-thymidine studies do indicate that when optimum concentration of these filarial proteins were added to lung cultures in proliferating and basal/maintenance media a further increase in growth stimulation was observed early in culture. However, on longer exposures and at higher concentrations an inhibitory effect with distinct morphological changes were noted. The dual role of these proteins on lung epithelial cells in vitro may highlight the possibility of a direct interaction of these proteins with lung cells during disease also contributing to tissue damage.

Isolation of microfilariae and eggs of Setaria digitata.

A simple procedure of gel-filtration was used to purify microfilariae of the cattle parasite, Setaria digitata from its eggs. The eggs of S. digitata were also isolated from the female worms by picking out the uterine canal. Gel-filtration gave a recovery of 70% for microfilariae.

Effect of diethylcarbamazine on acetylcholine and gamma amino butyric acid in Setaria digitata.

In vitro studies on the effect of neurotransmitter amino acids and amines on the motility of S. digitata showed that acetylcholine (Ach) had a stimulatory and gama amino butyric acid (GABA) an inhibitory effect on the parasite. When the worms were incubated in different concentrations of diethylcarbamazine there was a significant dose related increase in the level of Ach, and the level of GABA remained unchanged. Inhibition of acetylcholine esterase activity by diethylcarbamazine caused the accumulation of Ach in the synapses resulting in receptor desensitization and after a momentary stimulation causes paralysis of the parasite.