Adult Female Worms Research Articles

The optimal timing of post-treatment sampling for the assessment of anthelminthic drug efficacy against Ascaris infections in humans

The egg reduction rate (ERR) is the current standard mean to assess the efficacy of drugs against human soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura and hookworm). Although the timing of post-treatment sampling is pivotal for a readily interpretation of drug efficacy, there is lack empirical data that allows recommending the optimal time point for a follow-up egg counting. In the present study, we re-analyzed both the kinetics of worm expulsion and egg output for Ascaris lumbricoides following a single oral dose of albendazole in a series of studies previously conducted in Kenyan communities. The results indicate that it takes up to 10 days post-treatment before the expulsion of both adult male and female Ascaris worms is completed, approximately 20% of the worms being expelled between day 7 and 10 post-treatment. The sequential analysis of the egg out put, indicated a poor ERR (89.4%) at day 7 post-treatment, but a 100% ERR at day 14 and 21 post-treatment. Based on our findings we recommend to wait at least 14 days after an albendazole treatment before conducting the follow-up egg count. Any sampling before this time point may result in biased ERR estimates, due the release of residual eggs from moribund or degenerating worms.

Subconjunctival Loa Loa worm: A case report

Ocular Loiasis although not frequently encountered, is reported from various parts of India. We report one such case of subconjunctival loa loa worm in a patient, who had no history of international travel or immigration. A 62 year old male patient presented with complaints of pain, redness, irritation and watering in the left eye since one day. On examination of the left eye, there was conjunctival congestion and a white thread like mobile worm was noticed in the subconjunctival space nasally. Under aseptic precautions and topical anaesthesia, a single white 5 cm long worm was removed alive and sent for histopathological examination, which reported it as an adult female loa loa worm.

Histopathological Changes of Egg Cells in the Uterine of Ascaridia galli after Treatment with Extract of Veitchia merrillii Nuts

The aim of the study was to observe the histopathological changes of the entire egg cells in the uterine of Ascaridia galli worms treated with crude ethanolic extract of Veitchia merrillii nuts. Histopathological changes of the entire egg cells were observed to describe that the activity of extract V. merrillii nuts interfered the reproduction process could decrease the quantity and quality of A. galli eggs. The experiments consisted of 16 head A. galli female adult worms divided into four groups, contained four worms of each, and maintained in 0.9% phosphate buffered saline (PBS). First group, a negative control experiment consisted of A. galli in 0.9% PBS. Second group, concentration of 15 mg/mL albendazole was used as a positive control. Third group and fourth group, in vitro exposure of the worms to the crude ethanolic extract V. merrillii nuts at a concentration of 25 mg/mL and 75 mg/mL, respectively. The histopathological changes of egg cells in the uterine region of stained worms was evaluated by light microscopic examination at 40x magnification. The results showed that shrinkage and disintegration of the entire egg cells in the breakage of uterus. The results indicate that the possible use of the plant as a potential anthelmintic against A. galli, the intestinal nematode parasite of domestic fowl.

An ARV1 homologue from a filarial nematode is functional in yeast.

The transmembrane protein, ARV1, plays a key role in intracellular sterol homeostasis by controlling sterol distribution and cellular uptake. To date, only the ARV1s from yeast and humans have been characterized to some extent. In this study, the ARV1 of an animal filarial parasite, Setaria digitata (SdARV1), was characterized; its cDNA was 761 bp and encoded a protein of 217 amino acids, with a predicted molecular weight of 25 kDa, containing a highly conserved ARV1 homology domain and three transmembrane domains in the bioinformatic analyses. Information required to cluster members belonging to a particular taxon has been revealed in phylogenetic analyses of ARV1 sequences derived from different organisms. Reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that SdARV1 was expressed in different developmental stages - microfilariae and adult male and female worms. Experiments carried out with a single copy of the SdARV1 under the control of the PMA-1 promoter in a temperature-sensitive Saccharomyces cerevisiae mutant strain indicated full complementation of the mutant phenotype, with growth at a non-permissive temperature (37°C). Microscopic observations of cellular morphology with Gram staining revealed alteration of the shape from shrunken to oval, in mutant and complemented strains, respectively. Assessment of free sterol levels extracted from mutant yeast and complemented strains indicated that the level of sterol was significantly higher in the former compared to the latter, which had sterol levels similar to those of the wild type. Thus, the results of the current study suggest that SdARV1 is ubiquitously expressed in different developmental stages of S. digitata, and that it is a true functional homologue of mammalian and yeast ARV1s, which have crucial phylogenetic information that follows classical evolutionary trends. Finally, this is the first study to report the biological function of nematode ARV1.

Licochalcone A induces morphological and biochemical alterations in Schistosoma mansoni adult worms

This paper is the first report on the in vitro effects of licochalcone A, a chalcone isolated from Glycyrrhiza inflate Batalin (Leguminosae), on Schistosoma mansoni adult worms. In vitro, licochalcone A afforded lethal concentrations for 50% of parasites (LC50) of 9.12±1.1 and 9.52±0.9μM against female and male adult worms, respectively, at 24h. Additionally, the compound reduced the total number of S. mansoni eggs and affected the development of eggs produced by S. mansoni adult worms. Together, the results achieved after 24h showed that licochalcone A was 55.7- and 53.3-fold more toxic to S. mansoni female and male adult worms than to Chinese hamster ovary fibroblasts cells, respectively. Treatment with licochalcone A elicited drastic changes in the tegument of S. mansoni adult worms, as well as mitochondrial alteration and chromatin condensation. Licochalcone A also increased the superoxide anion level and decreased the superoxide dismutase activity in S. mansoni adult worms. Overall, our results indicated that licochalcone A displays in vitro schistosomicidal activity. This effect may result from increased production of reactive oxygen species (ROS) induced by the action of licochalcone A. The resulting ROS could act on the S. mansoni tegument and membranes and help induce the death of S. mansoni adult worms.

H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage.

ABSTRACTUrogenital schistosomiasis, caused by the parasitic trematode Schistosoma haematobium, affects over 112 million people worldwide. As with Schistosoma mansoni infections, the pathology of urogenital schistosomiasis is related mainly to the egg stage, which induces granulomatous inflammation of affected tissues. Schistosoma eggs and their secretions have been studied extensively for the related organism S. mansoni, which is more amenable to laboratory studies. Indeed, we have shown that IPSE/alpha-1 (here M-IPSE), a major protein secreted from S. mansoni eggs, can infiltrate host cells. Although the function of M-IPSE is unknown, its ability to translocate to the nuclei of host cells and bind DNA suggests a possible role in immune modulation of host cell tissues. Whether IPSE homologs are expressed in other schistosome species has not been investigated. Here, we describe the cloning of two paralog genes, H03-IPSE and H06-IPSE, which are orthologs of M-IPSE, from egg cDNA of S. haematobium. Using PCR and immunodetection, we confirmed that the expression of these genes is restricted to the egg stage and female adult worms, while the H-IPSE protein is detectable only in mature eggs and not adults. We show that both H03-IPSE and H06-IPSE proteins can infiltrate HTB-9 bladder cells when added exogenously to culture medium. Monopartite C-terminal nuclear localization sequence (NLS) motifs conserved in H03-IPSE, SKRRRKY, and H06-IPSE SKRGRKY, are responsible for targeting the proteins to the nucleus of HTB-9 cells, as demonstrated by site-directed mutagenesis and green fluorescent protein (GFP) tagging. Thus, S. haematobium eggs express IPSE homologs that appear to perform similar functions in infiltrating host cells.

Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity.

BackgroundTreatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana—exposed to more than a decade of regular ivermectin treatment—have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread.Methodology/Principal findingsPooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR.Conclusions/SignificanceThis study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations.

Immunologic evaluation of extracted intestinal proteins from Angiostrongylus cantonensis adult worms.

To determine whether intestinal Angiostrongylus cantonensis antigens can induce protective immunity in rats, gut antigens prepared from female adults (FAGP) and somatic antigens prepared from both male (MA) and female (FA) adult worms were used to immunize rats. Rats were immunized twice with MA, FA, or FAGP antigens and then challenged with 50 third-stage A. cantonensis larvae, and different readouts were used to monitor protective immunity. Additionally, protein profiles of MA, FA, and FAGP extracts were analyzed and characterized by immunodetection methods. A 15% reduction in fifth-stage larvae from brains and a 14% reduction in adult worms from pulmonary arteries were observed in rats immunized with FAGP compared to controls. However, there was a >50% reduction in rats immunized with MA or FA. The lengths of larvae and adults recovered from FAGP-immunized rats were shorter than those recovered from other groups. The number of first-stage larvae recovered from fecal material in FAGP-immunized rats was significantly reduced. Additionally, FAGP induced the highest splenocyte proliferation. Serum IgG titers were not directly correlated with protective immunity. An 84kDa gut membrane protein was strongly recognized by anti-FAGP antibodies. We demonstrated that immune responses induced by FAGP reduced the growth, development, and reproduction of A. cantonensis in subsequent infections. While the possibility of using FAGP combining with MA or FA antigens as a multi-function vaccine in immune protection against A. cantonensis needs to be further elucidated, we hope that it provides a novel strategy for this parasite vaccine development.

Fatty acid composition of free-living and parasitic stages of the bovine lungworm Dictyocaulus viviparus

The development of parasitic nematodes proceeds via multiple stages, often implicating the necessity to adapt to different environments. Especially the transition from free-living to parasitic stages is accompanied by a significant change in the environmental conditions. To shed light on possible adaptations to these transitions, the fatty acid composition of different developmental stages of the bovine lungworm Dictyocaulus viviparus was investigated. Fatty acids of D. viviparus eggs, the free-living first, second and third larval stage (L1–L3) as well as the parasitic preadult stage and adult male and female worms residing in the lungs of infected hosts were quantified by gas chromatography after transesterification to their fatty acid methyl esters. The fatty acid content and diversity were higher in parasitic stages compared to those of free-living larvae. The most prevalent fatty acids in both parasitic and free-living stages were stearic (C18:0), palmitic (C16:0), palmitoleic (C16:1) and caprylic acid (C8:0). A variety of (poly-)unsaturated FAs was found in the parasitic stages and in the eggs, which was similar to the variety of FAs found in bovine surfactant. This finding indicates that parasitic stages of D. viviparus take up FAs from their environment. While eggs contained the highest concentration of fatty acids, a decrease was observed from eggs to L1 and further from L2 to L3. The lowest concentration was found in 38-days-old L3, which suggests that FAs serve as an energy reserve for the free-living, non-feeding larval stages. The free-living larvae contained mainly saturated fatty acids and only traces of unsaturated fatty acids, which is in contrast to the phospholipid saturation hypothesis of cold tolerance. Instead, a trade-off between desiccation stress and temperature adaptation may favour a higher amount of saturated FAs in the free-living larval stages. Further studies explicitly examining the FA composition of the different classes of lipids are necessary to better describe the adaptative responses of the FA metabolism to different environmental conditions.

Angiostrongylus cantonensis: An optimized cultivation of this parasitic nematode under laboratory conditions.

Angiostrongylus cantonensis (A. cantonensis), a parasitic nematode, is the important neurotropic pathogen which causes human angiostrongyliasis. It has a complex life-cycle and severe parasite-host interaction in contrast to free-living nematode. Establishment of a well-suited life-cycle and in vitro cultivation of A. cantonensis in the laboratory will be one of the key techniques to elucidate the mechanism of parasite-host interaction. However, the low survival and growth rate of worms is still to be the problem. We optimized the known life-cycle of A. cantonensis in the laboratory, showing that small in size, easy to breed, and high compatibility of Biomphalaria straminea precede the common snails as an intermediate host of A. cantonensis. Furthermore, the egg hatching rate in Ham's F-12 medium reached approximately 80% using the eggs of mature female adult worms. We also demonstrated that the survival of larvae could be sustained for more than 30days by in vitro cultivation of L1 larvae in DMEM with mixed antibiotics (100units/mL of penicillin G potassium, 50μg/mL of streptomycin sulfate, and 0.5μg/mL of amphotericin B) and L3, L4, and L5 larvae in Waymouth's medium with 20% fetal calf serum and mixed antibiotics. Infective L1 and L3 larvae kept high infective rate to the snail and rat after cultivation in these media, respectively. It will provide the basis for studying on genetic manipulations for functional genes, new drug screening, and the mechanism of parasite-host interaction of parasitic nematodes.

Migratory phase of Litomosoides sigmodontis filarial infective larvae is associated with pathology and transient increase of S100A9 expressing neutrophils in the lung.

Filarial infections are tropical diseases caused by nematodes of the Onchocercidae family such as Mansonella perstans. The infective larvae (L3) are transmitted into the skin of vertebrate hosts by blood-feeding vectors. Many filarial species settle in the serous cavities including M. perstans in humans and L. sigmodontis, a well-established model of filariasis in mice. L. sigmodontis L3 migrate to the pleural cavity where they moult into L4 around day 9 and into male and female adult worms around day 30. Little is known of the early phase of the parasite life cycle, after the L3 is inoculated in the dermis by the vector and enters the afferent lymphatic vessels and before the moulting processes in the pleural cavity. Here we reveal a pulmonary phase associated with lung damage characterized by haemorrhages and granulomas suggesting L3 reach the lung via pulmonary capillaries and damage the endothelium and parenchyma by crossing them to enter the pleural cavity. This study also provides evidence for a transient inflammation in the lung characterized by a very early recruitment of neutrophils associated with high expression levels of S100A8 and S100A9 proteins.

Molecular characterization and transcriptional analysis of the female-enriched chondroitin proteoglycan 2 of Toxocara canis.

Toxocara canis is an important but neglected zoonotic parasite, and is the causative agent of human toxocariasis. Chondroitin proteoglycans are biological macromolecules, widely distributed in extracellular matrices, with a great diversity of functions in mammals. However, there is limited information regarding chondroitin proteoglycans in nematode parasites. In the present study, a female-enriched chondroitin proteoglycan 2 gene of T. canis (Tc-cpg-2) was cloned and characterized. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure the transcription levels of Tc-cpg-2 among tissues of male and female adult worms. A 485-amino-acid (aa) polypeptide was predicted from a continuous 1458-nuleotide open reading frame and designated as TcCPG2, which contains a 21-aa signal peptide. Conserved domain searching indicated three chitin-binding peritrophin-A (CBM_14) domains in the amino acid sequence of TcCPG2. Multiple alignment with the inferred amino acid sequences of Caenorhabditis elegans and Ascaris suum showed that CBM_14 domains were well conserved among these species. Phylogenetic analysis suggested that TcCPG2 was closely related to the sequence of chondroitin proteoglycan 2 of A. suum. Interestingly, a high level of Tc-cpg-2 was detected in female germline tissues, particularly in the oviduct, suggesting potential roles of this gene in reproduction (e.g. oogenesis and embryogenesis) of adult T. canis. The functional roles of Tc-cpg-2 in reproduction and development in this parasite and related parasitic nematodes warrant further functional studies.

Schistosoma mansoni displays an adenine phosphoribosyltransferase preferentially expressed in mature female gonads and vitelaria

Schistosoma mansoni depends upon the purine salvage pathway to obtain purine nucleotides; therefore, enzymes from this pathway are essential for parasite survival. Here, we focused on the adenine phosphoribosyltransferase (APRT) enzyme, which catalyzes the condensation reaction between adenine and PRPP (5-phosphoribosylpyrophosphate) to produce AMP and PPi. Kinetic experiments using the heterologously expressed protein of one APRT isoform from S. mansoni indicate that it is catalytically active, and whole-mount in situ hybridization studies indicate that the transcripts of this protein are concentrated in the posterior region of the ovary and vitellaria of female adult worms. Moreover, a phylogenetic analysis has shown that APRT exists in multiple copies originating from gene duplications at the base of the Schistosoma genus. Other enzymes from the purine and pyrimidine salvage pathways have also been found to present multiple copies in schistosomes, suggesting that evolutionary pressure to diversify these genes’ families may be related to a specialized role in parasite reproduction.

MODELING THE INFECTION DYNAMICS OF ONCHOCERCIASIS AND ITS TREATMENT

Onchocerciasis is one of the neglected tropical diseases caused by Onchocerca volvulus. Ivermectin is known to be effective in the treatment of onchocerciasis because it suppresses the production of microfilariae by the adult female worms for a few months following treatment thus reducing transmission. In this study, a deterministic model is developed to assess the effect of mass treatment of onchocerciasis with ivermectin. The basic reproduction number, [Formula: see text], of the model system is determined and it is observed that the model exhibits backward bifurcation for some parameters implying the existence of multiple endemic equilibria when [Formula: see text]. The existence of multiple equilibria emphasizes the fact that [Formula: see text] is not sufficient to eradicate the disease and the need is to lower [Formula: see text] much below one to make the disease-free equilibrium globally stable. Numerical simulations are done and conclusions drawn with respect to the known treatment protocols in endemic areas. The study results suggest that the mass treatment of the disease with ivermectin should cover a higher proportion of the population to control the disease and eventually eliminate it from the population.

SCANNING ELECTRON MICROSCOPIC CHANGES IN HABRONEMA MUSCAE AFTER IN VITRO EXPOSURE TO PLANT EXTRACT (VERBESINA ALTERNIFOLIA)

Scanning electron microscopy (SEM) was used to study the morphological alteration occurring in Habronema muscae adult female worms after in vitro exposure to different doses of Verbesina alternifolia oil extract. The half maximal lethal concentration (LC50) was reached 400 ppm after 24hrs, while LC100 was reached 600 ppm after 48hrs. Irreversible degenerative changes were recorded such as shrinking, detachment and distortion of the cuticle, cephalic and distal region. The cuticular surface had a wrinkled, corrugated appearance with longitudinal ridges and transverse thick folds. The lips and papillae were deformed and aggregated over each other. There is a direct relation between the level of the recorded degenerative changes and the increase in the dose and exposure time. In the same time no degen'erative changes were recorded in the control worm exposed to PBS till the end of the exposure period.

Tissue distribution and functional analysis of vitellogenin-6 of Toxocara canis

Toxocara canis is an common intestinal nematode of canids and the principal causative agent of human toxocariasis. Vitellogenin (Vg), a source of amino acids and lipids in the eggs, are considered to play an important role in embryo development of a wide range of organisms. In the present study, the transcriptional levels of Tc-vit-6 gene in male and female adult T. canis were determined by quantitative real-time PCR, which indicated high transcription of Tc-vit-6 in the intestine, reproductive tract and body wall of male and female adult T. canis. The fragment of Tc-vit-6 encoding a vWD domain, was cloned and expressed to produce a rabbit anti-TcvWD polyclonal antibody. Tissue distribution of TcVg6 was detected by immunohistochemical assays, which showed predominant distribution of TcVg6 in the tissues of intestine, as well as reproductive tract (including some of the germ cells) and musculature of male and female adult worms. Collectively, these results indicated multiple biological roles of TcVg6 apart from that in the reproduction of T. canis.

Oleanolic acid from antifilarial triterpene saponins of Dipterocarpus zeylanicus induces oxidative stress and apoptosis in filarial parasite Setaria digitata in vitro

Absence of a drug that kills adult filarial parasites remains the major challenge in eliminating human lymphatic filariasis (LF); the second leading cause of long-term and permanent disability. Thus, the discovery of novel antifilarial natural products with potent adulticidal activity is an urgent need. In the present study, methanol extracts of leaves, bark and winged seeds of Dipterocarpus zeylanicus (Dipterocarpaceae) were investigated for macro and microfilaricidal activity. Two antifilarial triterpene saponins were isolated from winged seed extracts by bioactivity guided chromatographic separation and identified using Nuclear Magnetic Resonance and mass spectroscopic analysis as oleanolic acid 3-O-β-D- glucopyranoside (1) (IC50 = 20.54 μM for adult worms, 19.71 μM for microfilariae ) and oleanolic acid 3-O-α-L-arabinopyranoside (2) (IC50 = 29.02 μM for adult worms, 25.99 μM for microfilariae). Acid hydrolysis of both compounds yielded oleanolic acid (3) which was non or least toxic to human peripheral blood mono nuclear cells (Selectivity index = >10) while retaining similar macrofilaricidal (IC50 = 38.4 μM) and microfilaricidal (IC50 = 35.6 μM) activities. In adult female worms treated with 50 and 100 μM doses of oleanolic acid, condensation of nuclear DNA, apoptotic body formation and tissue damage was observed by using Hoechst 33342 staining, TUNEL assay and Hematoxylin and Eosin staining respectively. A dose dependent increase in caspase 3/CED3 activity and decrease in total protein content were also observed in these parasites. A dose dependant DNA fragmentation was observed in adult parasites and microfilariae. Decreased levels of reduced glutathione (GSH) and elevated levels of glutathione S transferase (GST), superoxide dismutase (SOD) and reactive oxygen species (ROS) were also observed in parasites treated with oleanolic acid indicating an oxidative stress mediated apoptotic event. Compound 3/oleanolic acid was thus identified as a potent and safe antifilarial compound in vitro.

Modulation of Haemonchus contortus infection by depletion of γδ+ T cells in parasite resistant Canaria Hair Breed sheep

Canaria Hair Breed (CHB) sheep display resistance against the adult stage of the nematode, Haemonchus contortus. Previous studies have suggested significant correlations between γδ+ T lymphocytes and fecundity of female adult worms, suggesting a novel role in immune modulation by these cells. The largest proportion of γδ+ T lymphocytes in sheep are the subpopulation of γδ+/WC1+ T cells. The aim of this study was to evaluate the effect of γδ+⁄WC1+ T cell depletion via infusion of anti-γδ/WC1 monoclonal antibody (mAb) on the subsequent immune response of CHB sheep infected with H. contortus. Significantly lower γδ+ T cell levels in both peripheral blood and in the basal layers of the abomasal tissue resulted following anti-γδ/WC1 mAb infusion of CHB sheep compared to control animals. Worms recovered from the anti-γδ/WC1 mAb treated CHB sheep had significantly longer female worms with correspondingly more eggs in utero than the saline control group. Significant correlations between eosinophils and worm length and fecundity were no longer apparent in the anti-γδ/WC1 mAb treated CHB sheep. These results support the notion that γδ+ T cells in CHB sheep play a critical role in fecundity regulation (length and eggs in utero) of H. contortus adult female worms, and highlights a new mechanism of modulation by this lymphocyte population, possibly involving eosinophil activation.

Development of an in Vitro RNAi to Investigate Gene Function in Brugia Malayi

The RNA interference (RNAi) has the ability to turn off individual gene expression. So, it affords a remarkably specific tool for studying the effects of genes. It is regarded as a direct approach for determining such gene/genes functions and offers a valuable tool for modern drug discovery. The study aimed to develop in vitro RNAil in Brugia malayi with particular interest to study the function of Brugia malayi avr-14 (Bm-avr-14) and Brugia malayi f-tubulin (Bm-fi-tubulin) genes. Bm-avr-14 is a gene encoding glutamate gated chloride channel (GluCl) which binds ivermectin and Bm-β-tubulin is a gene encoding β-tubulin which binds albendazole. Adult female worms were soaked in heterogeneous short interfering RNA (hsiRNA) with interest to study the role of two genes Bm-avr-14 and Bm-β-tubulin. Then, we assessed the knock down effects of target genes using worminator system and real time PCR. We found that worms treated with hsiRNA of Bm-avr-14 had a significant reduction in microfilariae (mf) production in comparison with untreated worms or worms treated with hsiRNA of green fluorescent protein (GFP). No significant reduction in mf production with Bm-β-tubulin gene was obtained. There were no changes in the movement of adults treated with either Bm-avr-14 or Bm- β-tubulin hsiRNAs. Inconsistent RNAi mediated suppression was achieved with Bm-avr-14 and Bm-β- tubulin using real time PCR. The data may be helpful in assessment of drug target potential of genes.

DEVELOPMENT OF AN IN VITRO RNAI TO INVESTIGATE GENE FUNCTION IN BRUGIA MALAYI

The RNA interference (RNAi) has the ability to turn off individual gene expression. So, it affords a remarkably specific tool for studying the effects of genes. It is regarded as a direct approach for determining such gene/genes functions and offers a valuable tool for modern drug discovery. The study aimed to develop in vitro RNAil in Brugia malayi with particular interest to study the function of Brugia malayi avr-14 (Bm-avr-14) and Brugia malayi f-tubulin (Bm-fi-tubulin) genes. Bm-avr-14 is a gene encoding glutamate gated chloride channel (GluCl) which binds ivermectin and Bm-β-tubulin is a gene encoding β-tubulin which binds albendazole. Adult female worms were soaked in heterogeneous short interfering RNA (hsiRNA) with interest to study the role of two genes Bm-avr-14 and Bm-β-tubulin. Then, we assessed the knock down effects of target genes using worminator system and real time PCR. We found that worms treated with hsiRNA of Bm-avr-14 had a significant reduction in microfilariae (mf) production in comparison with untreated worms or worms treated with hsiRNA of green fluorescent protein (GFP). No significant reduction in mf production with Bm-β-tubulin gene was obtained. There were no changes in the movement of adults treated with either Bm-avr-14 or Bm- β-tubulin hsiRNAs. Inconsistent RNAi mediated suppression was achieved with Bm-avr-14 and Bm-β- tubulin using real time PCR. The data may be helpful in assessment of drug target potential of genes.