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Sample records for brugia

  1. Transient effects of levamisole on Brugia malayi microfilariae.

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    Mostafa, Eman; Storey, Bob; Farghaly, Adel Mohammed; Afify, Hany Abd El-Hai; Taha, Afaf Abd El-Raouf; Wolstenholme, Adrian J

    2015-09-01

    Levamisole is an anthelmintic drug that acts by activating nicotinic acetylcholine receptors at the nematode neuromuscular junction and causing paralysis. We measured the in vitro effects of levamisole on the motility of Brugia malayi microfilariae; after 2 h incubation the apparent IC50 was 2.68 mM. Lower drug concentrations, such as 1 mM, caused an immediate total paralysis that lasted for up to 1 h, but was completely reversed by 2 h of incubation. The 'recovered' parasites were still completely susceptible to application of a second nicotinic agonist, pyrantel.

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

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    Ciaran J. McCoy; Reaves, Barbara J.; Giguère, Steeve; Coates, Ruby; Rada, Balázs; Wolstenholme, Adrian J.

    2017-01-01

    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 expos...

  3. Draft genome of the filarial nematode parasite Brugia malayi.

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    Ghedin, Elodie; Wang, Shiliang; Spiro, David; Caler, Elisabet; Zhao, Qi; Crabtree, Jonathan; Allen, Jonathan E; Delcher, Arthur L; Guiliano, David B; Miranda-Saavedra, Diego; Angiuoli, Samuel V; Creasy, Todd; Amedeo, Paolo; Haas, Brian; El-Sayed, Najib M; Wortman, Jennifer R; Feldblyum, Tamara; Tallon, Luke; Schatz, Michael; Shumway, Martin; Koo, Hean; Salzberg, Steven L; Schobel, Seth; Pertea, Mihaela; Pop, Mihai; White, Owen; Barton, Geoffrey J; Carlow, Clotilde K S; Crawford, Michael J; Daub, Jennifer; Dimmic, Matthew W; Estes, Chris F; Foster, Jeremy M; Ganatra, Mehul; Gregory, William F; Johnson, Nicholas M; Jin, Jinming; Komuniecki, Richard; Korf, Ian; Kumar, Sanjay; Laney, Sandra; Li, Ben-Wen; Li, Wen; Lindblom, Tim H; Lustigman, Sara; Ma, Dong; Maina, Claude V; Martin, David M A; McCarter, James P; McReynolds, Larry; Mitreva, Makedonka; Nutman, Thomas B; Parkinson, John; Peregrín-Alvarez, José M; Poole, Catherine; Ren, Qinghu; Saunders, Lori; Sluder, Ann E; Smith, Katherine; Stanke, Mario; Unnasch, Thomas R; Ware, Jenna; Wei, Aguan D; Weil, Gary; Williams, Deryck J; Zhang, Yinhua; Williams, Steven A; Fraser-Liggett, Claire; Slatko, Barton; Blaxter, Mark L; Scott, Alan L

    2007-09-21

    Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the approximately 90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict approximately 11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during approximately 350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.

  4. Brugia timori INFECTION IN LEKEBAI, FLORES: clinical aspects

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    Arbain Joesoef

    2012-09-01

    Full Text Available Pengamatan filariasis pada penduduk Nualolo-Lekebai, Pulau Flores telah dilakukan pada bulan Februari 1975. Kampung Nualolo-Lekebai berpenduduk 680 jiwa, pekerjaan bertani dan menganut agama Nasrani. Kebiasaan hidup di antara penduduk di daerah ini adalah menyerahkan pelaksanaan pekerjaan berat pada kaum wanita, baik di rumah ataupun di kebun. Dalam perjalanan jauh baik ke kebun atau ke pasar, kaum wanitanya selalu berjalan kaki sedangkan kaum prianya menunggang kuda. Sejumlah 80% dari penduduk kampung ini telah diperiksa terhadap infeksi parasit filaria dan terhadap gejala filariasis. Dari hasil yang ditemukan ternyata penduduk kampung ini menderita infeksi Brugia timori dengan angka derajat infeksi sebesar 7.0% dan angka derajat elephantiasis sebesar 10.3%. Hal yang menarik yang ditemukan dalam pengamatan ini adalah tingginya angka derajat elephantiasis pada kaum wanita dibandingkan dengan pada kaum pria. Fenomena ini mungkin disebabkan oleh kebiasaan hidup kaum wanita di daerah ini sehari-hari yang bekerja lebih berat dan berjalan kaki lebih sering dan lebih jauh dibandingkan kaum prianya.

  5. Detection of filaria-specific IgG4 antibodies using Brugia Rapid test in individuals from an area highly endemic for Brugia timori.

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    Supali, T; Rahmah, N; Djuardi, Y; Sartono, E; Rückert, Paul; Fischer, P

    2004-05-01

    The filarial parasite Brugia timori is of great public health importance in some islands of Eastern Indonesia. To establish a simple serological test for the identification and post-treatment monitoring of areas endemic for B. timori, a rapid immunochromatographic dipstick test (Brugia Rapid, BR) was evaluated on microfilaraemic and amicrofilaraemic individuals. This test is based on the detection of anti-filarial IgG4 antibodies that react with a recombinant Brugia malayi antigen (BmR1). In our study area on Alor island the prevalence of microfilaraemia was 26%. With the BR test, 100% of 196 sera from microfilaraemic persons and 76% of 563 sera from amicrofilaraemic persons, either symptomatic or asymptomatic, reacted positive. All 50 control sera from areas non-endemic for lymphatic filariasis gave negative BR test results. This study showed that the BR test can be also used to detect antibodies against B. timori. Due to the high prevalence of IgG4 antibodies as detected by the BR test (81%), no significant correlation with the prevalence of microfilaraemia could be detected within the endemic village. The BR test also shows great promise to be employed as a monitoring tool for B. timori in the framework of the Global Program to Eliminate Lymphatic Filariasis (GPELF).

  6. Ultrastructural studies on the microfilaria of Brugia malayi

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    Tongu,Yasumasa

    1974-06-01

    Full Text Available Ultrastructure of microfilaria Brugia malayi was investigated with electron microscope. Microfilariae are covered by a sheath membrane with dense materials on its outer surfaces. The cuticle consists of 3 layers; namely, external cortical, internal cortical and fibrous layer. Beneath these cuticular layers, thin hypodermis is present and the muscle cells are arranged of 4 groups in a crosssection except for the head and tail. A pair of cephalic channel containing several cilial rods opens at the anterior end of the worm. A hook is situated on the anterior edge of one channel orifice, and several spines grow on the opposite side to the hook. Caudal channels paired laterally opening into the both sides of the posterior region differ from cephalic channels by the presence of a single cilial rod. A central canal runs from the buccal cavity to the inner body, and opens into the inner body cell through the filamentous apparatus. The inner body appears to consist of several cells having storage substances and a flat nucleus located on the periphery of the cell. An excretory apparatus, i. e., a cell, is composed of a nucleus and a large vesicle which has many microprojections on the luminal surfaces. The GI cell which occupies the whole width in a cross-section is larger than the R cell. R2-R4 cells appear to be in a close contact with the anal apparatus having many microprojections on the luminal surfaces. These microprojections differ from those of the excretory vesicle in their thickness and length. The characteristic patterns of these organs are compared with other microfilariae.

  7. Effects of gamma radiation on development of Brugia pahangi in a susceptible strain of Aedes aegypti

    Energy Technology Data Exchange (ETDEWEB)

    Richey, T.J.; Rodriguez, P.H.

    1976-08-01

    Female mosquitoes were fed on an infected jird having a microfilarial density of 201 per mm/sup 3/ of blood. Mosquitoes were exposed to doses of 3,000 and 5,000 rads of gamma radiation before and after infection. Mosquitoes were dissected 8 to 10 days later and the number of active larvae was recorded. Results indicated that postinfection radiation affected the development of Brugia pahangi. (HLW)

  8. Detection of Brugia malayi microfilaria/Larvae in mosquito using Polimerase Chain Reaction.

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    Dyah Haryuningtyas

    2008-10-01

    Full Text Available Lymphathic filariasis that is also known as elepanthiasis is caused by infestation of 3 species nematode Wuchereria bancrofti, Brugia malayi and Brugia timori. In Indonesia 70% filariasis case caused by Brugia malayi. Mosquito species from genus Anopheles, Aedes, Culex, Mansonia and Armigeres are known as vector of this disease. Microfilaria detection on mosquito is one methode to know infection rate in vector population in endemic area.The objectives of the research were to study the ability of Hha1 repeat applicable to detect microfilaria/larvae in a pool of mosquitoes and to get description of adult mosquito night biting population lived in endemic area of filariasis brugian. Mosquito as positive control used in this research come from laboratory of parasitology of FKUI. Mosquito sample from the field was from Binawara and Kolam Kiri villages, South Kalimantan province. Mosquito were trapped then identified by its species. DNA of mosquitoes was extracted and then run by the PCR using Hha 1 repeat primer. Result of the research indicated that adult mosquitoes night biting from Binawara village consist of Culex, Mansonia, Anopheles genus and from Kolam Kiri village only from Mansonia genus. Hha 1 repeat primer is applicable to detect 1 mosquito infected with microfilaria/larvae in a pool of negative mosquitoes. Mosquito samplesfrom the two villages showing negative PCR.

  9. HYBRIDOMA PRODUCTION USING IMMUNE LYMPHOCYTES AGAINST BRUGIA MALAYI ANTIGEN WITH MYELOMA CELLS

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    Soeyoko Soeyoko

    2012-09-01

    Full Text Available Wuchereria bancrofti, Brugia malayi, and Brugia timori are the causative agents of lymphatic hiariasis in Indonesia, but in some endemic areas, Brugia malayi is the most commonly found,Diagnosis of hiariasis is normally based on clinical and parasitological examinations, but both have limitations. Therefore now the immunological examination plays an important role in the diagnosis of filariasis. The discovery of monoclonal antibodies recently may probably give a firm scientific basis in immunology and add a new dimension to the efforts of developing a specific and sensitive immunological test for various stages of filarial infection. In this study, the production of hybridoma cells to develop monoclonal antibodies against B. malayi integrated a number of techniques: preparations of B. malayi surface antigen, immune lymphocyte cells, NSI myeloma cells and macrophage feeder layers, and a fusion of immune lymphocytes with myeloma cells. The results of this study can be concluded in three points: Protein analysis of the surface antigen was examined by Biureet and SDS-PAGE. A total of fourteen examinations were conducted by using 4000 L3 for each experiment. Three were not detected by Biureet method, but showed five protein fractions by SDS-PAGE. The protein concentration of the surface L3 was varied from 85.0/µg/ml to 769.23/µg/ml, with an average of 297.04/µg/ml.The immunoreactivifies of Balb/c mice antibodies to B. malayi L3 surface antigen were tested by ELISA and showed a gradual increase after four times immunizations at two weeks interval. The optical density (OD after four times immunizations was varied from 0.363 to 0.878 each mouse, where as the positive control sera OD was 0.570.Hybridization using immune lymphocytes against B. malayi L3 surface antigen with myeloma cells yielded 60.41% hybrid cells and none of them produced monoclonal antibodies tested of ELISA.

  10. Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi.

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    Elizabeth Bilsland

    2016-01-01

    Full Text Available Lymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7-15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases.We have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts, and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds, we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi.We were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-based screening platform is efficient in identifying compounds that can discriminate between human and filarial enzymes. Hence, we are confident

  11. In vitro antifilarial effects of three plant species against adult worms of subperiodic Brugia malayi.

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    Zaridah, M Z; Idid, S Z; Omar, A W; Khozirah, S

    2001-11-01

    Five aqueous extracts from three plant species, i.e., dried husks (HX), dried seeds (SX) and dried leaves (LX) of Xylocarpus granatum (Meliaceae), dried stems (ST) of Tinospora crispa (Menispermaceae) and dried leaves (LA) of Andrographis paniculata (Acanthaceae) were tested in vitro against adult worms of subperiodic Brugia malayi. The relative movability (RM) value of the adult worms over the 24-h observation period was used as a measure of the antifilarial activity of the aqueous extracts. SX extract of X. granatum demonstrated the strongest activity, followed by the LA extract of A. paniculata, ST extract of T. crispa, HX extract and LX extract of X. granatum.

  12. The Structure and Dynamics of BmR1 Protein from Brugia malayi: In Silico Approaches

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    Bee Yin Khor

    2014-06-01

    Full Text Available Brugia malayi is a filarial nematode, which causes lymphatic filariasis in humans. In 1995, the disease has been identified by the World Health Organization (WHO as one of the second leading causes of permanent and long-term disability and thus it is targeted for elimination by year 2020. Therefore, accurate filariasis diagnosis is important for management and elimination programs. A recombinant antigen (BmR1 from the Bm17DIII gene product was used for antibody-based filariasis diagnosis in “Brugia Rapid”. However, the structure and dynamics of BmR1 protein is yet to be elucidated. Here we study the three dimensional structure and dynamics of BmR1 protein using comparative modeling, threading and ab initio protein structure prediction. The best predicted structure obtained via an ab initio method (Rosetta was further refined and minimized. A total of 5 ns molecular dynamics simulation were performed to investigate the packing of the protein. Here we also identified three epitopes as potential antibody binding sites from the molecular dynamics average structure. The structure and epitopes obtained from this study can be used to design a binder specific against BmR1, thus aiding future development of antigen-based filariasis diagnostics to complement the current diagnostics.

  13. Characterization of innate immunity genes in the parasitic nematode Brugia malayi.

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    Libro, Silvia; Slatko, Barton E; Foster, Jeremy M

    The filarial nematode Brugia malayi is one of the causative agents of lymphatic filariasis, a neglected tropical disease that affects 120 million people worldwide. The limited effectiveness of available anthelmintics and the absence of a vaccine have prompted extensive research on the interaction between Brugia and its obligate bacterial endosymbiont, Wolbachia. Recent studies suggest that Wolbachia is able to manipulate its nematode host immunity but relatively little is known about the immune system of filarial nematodes. Therefore, elucidation of the mechanisms underlying the immune system of B. malayi may be useful for understanding how the symbiotic relationship is maintained and help in the identification of new drug targets. In order to characterize the main genetic pathways involved in B. malayi immunity, we exposed adult female worms to two bacterial lysates (Escherichia coli and Bacillus amyloliquefaciens), dsRNA and dsDNA. We performed transcriptome sequencing of worms exposed to each immune elicitor at two different timepoints. Gene expression analysis of untreated and immune-challenged worms was performed to characterize gene expression patterns associated with each type of immune stimulation. Our results indicate that different immune elicitors produced distinct expression patterns in B. malayi, with changes in the expression of orthologs of well-characterized C. elegans immune pathways such as insulin, TGF-β, and p38 MAPK pathways, as well as C-type lectins and several stress-response genes.

  14. Molecular evidence for a functional ecdysone signaling system in Brugia malayi.

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    George Tzertzinis

    Full Text Available BACKGROUND: Filarial nematodes, including Brugia malayi, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor and USP (Ultraspiracle. METHODS AND FINDINGS: We report the cloning and characterization of a B. malayi EcR homologue (Bma-EcR. Bma-EcR dimerizes with insect and nematode USP/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE. In support of the existence of an active ecdysone receptor in Brugia we also cloned a Brugia rxr (retinoid X receptor homolog (Bma-RXR and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic B. malayi embryos transfected with a luciferase construct controlled by an EcRE engineered in a B. malayi promoter, in the presence of 20-hydroxy-ecdysone. CONCLUSIONS: Our study identifies and characterizes the two components (Bma-EcR and Bma-RXR necessary for constituting a functional ecdysteroid receptor in B. malayi. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live B. malayi embryos. These results together

  15. Enzootic Brugia infection in a two-year-old Colombian girl.

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    Kozek, W J; Reyes, M A; Ehrman, J; Garrido, F; Nieto, M

    1984-01-01

    Mature male and female filarial worms were present in a lymphatic vessel near a retro-auricular lymph node removed from a 2-year old Colombian girl. Although apparently mature microfilariae were present in the uterus of the female, none was detected in blood samples (Knott's) of peripheral blood taken during day or night. The identification of the worms as a species of Brugia is suggested by their location in a dilated lymphatic vessel, morphology of adults, and the distribution pattern of tail nuclei of the microfilariae. This case is similar to another Colombian case reported from the Departamento del Santander del Sur, and suggests that zoonotic filarial infections may not be uncommon in Colombia.

  16. In vitro biological evaluation of biguanides and dihydrotriazines against Brugia malayi and folate reversal studies.

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    Bag, Seema; Tawari, Nilesh R; Sharma, Richa; Goswami, Kalyan; Reddy, M V R; Degani, Mariam S

    2010-01-01

    Dihydrofolate reductase (DHFR) is a well-known target for antibacterial and anticancer therapy. DHFR inhibitors are useful for protozoan parasites, but are yet to be explored against metazoan species; hence the present work was designed to evaluate the efficacy of DHFR inhibitors against filariasis, one of the major neglected tropical diseases. Molecules from our in-house library of synthetic antifolate agents (biguanide and dihydrotriazine derivatives) were evaluated along with the antimalarial drug pyrimethamine and the antibacterial drug trimethoprim in an in vitro model against Brugia malayi microfilariae (Mf). Three biguanides and two dihydrotriazines were more potent than trimethoprim and pyrimethamine against B. malayi Mf. Trimethoprim, pyrimethamine and four of the five compounds active against Mf were also active against adult worms. To probe the mechanism of action of the compounds, reversal of activity of active compounds by folic acid and folinic acid was studied. In conclusion, DHFR inhibitors could be used as leads for new antifilarial drugs.

  17. The Wolbachia endosymbiont of Brugia malayi has an active pyruvate phosphate dikinase.

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    Raverdy, Sylvine; Foster, Jeremy M; Roopenian, Erica; Carlow, Clotilde K S

    2008-08-01

    Genome analysis of the glycolytic/gluconeogenic pathway in the Wolbachia endosymbiont from the filarial parasite Brugia malayi (wBm) has revealed that wBm lacks pyruvate kinase (PK) and may instead utilize the enzyme pyruvate phosphate dikinase (PPDK; ATP:pyruvate, orthophosphate phosphotransferase, EC 2.7.9.1). PPDK catalyses the reversible conversion of AMP, PPi and phosphoenolpyruvate (PEP) into ATP, Pi and pyruvate. The glycolytic pathway of most organisms, including mammals, contains exclusively PK for the production of pyruvate from PEP. Therefore, the absence of PPDK in mammals makes the enzyme an attractive Wolbachia drug target. In the present study, we have cloned and expressed an active wBm-PPDK, thereby providing insight into the energy metabolism of the endosymbiont. Our results support the development of wBm-PPDK as a promising new drug target in an anti-symbiotic approach to controlling filarial infection.

  18. Effects of Doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo

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    Rao Ramakrishna U

    2012-02-01

    Full Text Available Abstract Background Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi. Methods Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles. Results and discussion Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%. In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes. Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion. Conclusions Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron

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

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    McCoy, Ciaran J.; Reaves, Barbara J.; Giguère, Steeve; Coates, Ruby; Rada, Balázs

    2017-01-01

    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

  20. Antifilarial activity of Zoanthus species (Phylum Coelenterata, Class Anthzoa) against human lymphatic filaria, Brugia malayi.

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    Lakshmi, V; Saxena, A; Pandey, K; Bajpai, Preeti; Misra-Bhattacharya, Shailja

    2004-07-01

    The chloroform methanol (1:1) extract of an unidentified green zoanthus (Phylum Coelenterata, Class Anthozoa) showed promising in vitro adulticidal activity with a lethal concentration of 125 microg/ml on Brugia malayi. This extract brought about a 52.2% reduction in circulating microfilariae of B. malayi when administered to infected Mastomys coucha at 250 mg/kg, orally for 5 consecutive days. Further fractionation of the extract led to the recovery of four fractions, which were evaluated simultaneously in both in vitro and in vivo systems against B. malayi. The chloroform fraction at 250 mg/kg orally for 5 days exhibited the highest macrofilaricidal action (42.5%), closely followed by the insoluble n-butanol fraction (34.3%), the soluble hexane fraction (32.4%), and the soluble n-butanol fraction (20.4%). In addition, the hexane soluble fraction caused 44.3% sterilization of the surviving female parasites. Two compounds isolated were found devoid of antifilarial activity.

  1. Anti-idiotypic antibodies function as a surrogate surface epitope of Brugia malayi infective larvae.

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    Carlow, C K; Busto, P; Storey, N; Philipp, M

    1990-07-01

    Anti-idiotypic (AB2) antibodies were generated in rabbits following immunization with a murine IgM monoclonal antibody (AB1) recognizing a surface determinant of Brugia malayi infective stage larvae. AB2 specifically inhibited the binding of AB1 to B. malayi larvae. Furthermore, AB2 had the ability to mimic the original antigen since mice immunized with AB2 possessed serum antibodies (AB3) specific for the B. malayi surface determinant. The presence of anti-surface antibodies (AB3 and AB1) induced either by AB2 immunization or by administration of AB1, did not alter the outcome of an intraperitoneal infection of B. malayi larvae in BABL/c mice when compared to untreated animals. AB3 antibodies like AB1, were IgM, thus indicating an isotype restricted response to the B. malayi epitope. There were no detectable cell mediated responses to the surface determinant in mice immunized with AB2, assessed by lymphocyte blastogenesis or IL3 production in vitro in response to the idiotope as presented by living larvae. The lack of cellular responses and/or the previously demonstrated rapid shedding of the epitope may explain the inability of AB1 or AB2 to protect mice against larval challenge in this study.

  2. Antifilarial activity of gum from Moringa oleifera Lam. on human lymphatic filaria Brugia malayi

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    V Kushwaha

    2011-01-01

    Full Text Available Aim: Currently available antifilarial drugs diethylcarbamazine, ivermectin and albendazole and their combinations, are not able to control lymphatic filariasis. Therefore, a better antifilarial agent is urgently required for proper management of the disease. Materials and Methods: In this study, we evaluated the antifilarial activity of gum extract of plant Moringa oleifera Lam. against the human lymphatic filarial parasite Brugia malayi using adult worms and microfilariae (mf in two in vitro assays (motility and inhition in MTT reduction for viability and two animal models, primary (Meriones unguiculatus implanted with B. malayi adult worms in the peritoneal cavity and secondary (subcutaneous B. malayi infective larvae induced Mastomys coucha, the model closer to the natural human filarial infection screens. Results: The gum extract inhibited 100% motility (irreversible loss of motility of mf and inhibited more than 56% MTT reduction potential of the adult female worms. The extract was safe in cytotoxicity test using Vero cell line, therefore followed in vivo in primary and secondary screens. In primary screen, the extract (5×500 mg/kg caused 69% macrofilaricidal and 83% sterilization of female worms and 44% macrofilaricidal activity in secondary screen (5 × 1000 mg/kg by oral route. Conclusion: Thus, it is concluded that the gum of the plant is macrofilaricidal in both in vitro and in vivo and may provide valuable leads for design and development of new antifilarial agents. This is the first ever report on the antifilarial efficacy of M. oleifera.

  3. Diversity and expression of microRNAs in the filarial parasite, Brugia malayi.

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    Catherine B Poole

    Full Text Available Human filarial parasites infect an estimated 120 million people in 80 countries worldwide causing blindness and the gross disfigurement of limbs and genitals. An understanding of RNA-mediated regulatory pathways in these parasites may open new avenues for treatment. Toward this goal, small RNAs from Brugia malayi adult females, males and microfilariae were cloned for deep-sequencing. From ∼ 30 million sequencing reads, 145 miRNAs were identified in the B. malayi genome. Some microRNAs were validated using the p19 RNA binding protein and qPCR. B. malayi miRNAs segregate into 99 families each defined by a unique seed sequence. Sixty-one of the miRNA families are highly conserved with homologues in arthropods, vertebrates and helminths. Of those miRNAs not highly conserved, homologues of 20 B. malayi miRNA families were found in vertebrates. Nine B. malayi miRNA families appear to be filarial-specific as orthologues were not found in other organisms. The miR-2 family is the largest in B. malayi with 11 members. Analysis of the sequences shows that six members result from a recent expansion of the family. Library comparisons found that 1/3 of the B. malayi miRNAs are differentially expressed. For example, miR-71 is 5-7X more highly expressed in microfilariae than adults. Studies suggest that in C.elegans, miR-71 may enhance longevity by targeting the DAF-2 pathway. Characterization of B. malayi miRNAs and their targets will enhance our understanding of their regulatory pathways in filariads and aid in the search for novel therapeutics.

  4. Diversity and expression of microRNAs in the filarial parasite, Brugia malayi.

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    Poole, Catherine B; Gu, Weifeng; Kumar, Sanjay; Jin, Jingmin; Davis, Paul J; Bauche, David; McReynolds, Larry A

    2014-01-01

    Human filarial parasites infect an estimated 120 million people in 80 countries worldwide causing blindness and the gross disfigurement of limbs and genitals. An understanding of RNA-mediated regulatory pathways in these parasites may open new avenues for treatment. Toward this goal, small RNAs from Brugia malayi adult females, males and microfilariae were cloned for deep-sequencing. From ∼ 30 million sequencing reads, 145 miRNAs were identified in the B. malayi genome. Some microRNAs were validated using the p19 RNA binding protein and qPCR. B. malayi miRNAs segregate into 99 families each defined by a unique seed sequence. Sixty-one of the miRNA families are highly conserved with homologues in arthropods, vertebrates and helminths. Of those miRNAs not highly conserved, homologues of 20 B. malayi miRNA families were found in vertebrates. Nine B. malayi miRNA families appear to be filarial-specific as orthologues were not found in other organisms. The miR-2 family is the largest in B. malayi with 11 members. Analysis of the sequences shows that six members result from a recent expansion of the family. Library comparisons found that 1/3 of the B. malayi miRNAs are differentially expressed. For example, miR-71 is 5-7X more highly expressed in microfilariae than adults. Studies suggest that in C.elegans, miR-71 may enhance longevity by targeting the DAF-2 pathway. Characterization of B. malayi miRNAs and their targets will enhance our understanding of their regulatory pathways in filariads and aid in the search for novel therapeutics.

  5. Mining predicted essential genes of Brugia malayi for nematode drug targets.

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    Sanjay Kumar

    Full Text Available We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression.

  6. Transcription profiling reveals stage- and function-dependent expression patterns in the filarial nematode Brugia malayi

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    Li Ben-Wen

    2012-05-01

    Full Text Available Abstract Background Brugia malayi is a nematode parasite that causes lymphatic filariasis, a disfiguring and disabiling tropical disease. Although a first draft genome sequence was released in 2007, very little is understood about transcription programs that govern developmental changes required for the parasite’s development and survival in its mammalian and insect hosts. Results We used a microarray with probes that represent some 85% of predicted genes to generate gene expression profiles for seven parasite life cycle stages/sexes. Approximately 41% of transcripts with detectable expression signals were differentially expressed across lifecycle stages. Twenty-six percent of transcripts were exclusively expressed in a single parasite stage, and 27% were expressed in all stages studied. K-means clustering of differentially expressed transcripts revealed five major transcription patterns that were associated with parasite lifecycle stages or gender. Examination of known stage-associated transcripts validated these data sets and suggested that newly identified stage or gender-associated transcripts may exercise biological functions in development and reproduction. The results also indicate that genes with similar transcription patterns were often involved in similar functions or cellular processes. For example, nuclear receptor family gene transcripts were upregulated in gene expression pattern four (female-enriched while protein kinase gene family transcripts were upregulated in expression pattern five (male-enriched. We also used pair-wise comparisons to identify transcriptional changes between life cycle stages and sexes. Conclusions Analysis of gene expression patterns of lifecycle in B. malayi has provided novel insights into the biology of filarial parasites. Proteins encoded by stage-associated and/or stage-specific transcripts are likely to be critically important for key parasite functions such as establishment and maintenance of

  7. The secreted triose phosphate isomerase of Brugia malayi is required to sustain microfilaria production in vivo.

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    James P Hewitson

    2014-02-01

    Full Text Available Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI, a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60-70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4⁺ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections.

  8. Cross reactive molecules of human lymphatic filaria Brugia malayi inhibit Leishmania donovani infection in hamsters.

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    Verma, Richa; Joseph, Sujith K; Kushwaha, Vikas; Kumar, Vikash; Siddiqi, M I; Vishwakarma, Preeti; Shivahare, Rahul; Gupta, Suman; Murthy, P K

    2015-12-01

    Coinfections are common in natural populations and the outcome of their interactions depends on the immune responses of the host elicited by the parasites. Earlier we showed that immunization with BmAFII (Sephadex G-200 eluted) fraction of human lymphatic filaria Brugia malayi inhibited progression of Leishmania donovani infection in golden hamsters. In the present study we identified cross reactive molecules of B. malayi, and investigated their effect on L. donovani infection and associated immune responses in the host. The sequence alignment and sharing of linear T- and B-cell epitopes in protein molecules of B. malayi and L. donovani counterparts were studied in silico. Hamsters were immunized with robustly cross reactive SDS-PAGE resolved fractions F6 (54.2-67.8kDa) and F9 (41.3-45.0kDa) of B. malayi and subsequently inoculated with amastigotes of L. donovani intracardially. F6 inhibited (∼72%) L. donovani infection and upregulated Th1 cytokine expression, lymphoproliferation, IgG2, IgG2/3 levels and NO production, and downregulated Th2 cytokine expression. Sequences in HSP60 and EF-2 of F6 and L. donovani counterparts were conserved and B- and T-cell epitopes in the proteins shared antigenic regions. In conclusion, leishmania-cross reactive molecules of filarial parasite considerably inhibited leishmanial infection via Th1-mediated immune responses and NO production. Common B- and T-cell epitope regions in HSP60 and EF-2 of the parasites might have contributed to the inhibitory effect on the L. donovani infection. Thus, leishmania-cross reactive filarial parasite molecules may help in designing prophylactic(s) against L. donovani.

  9. Mining predicted essential genes of Brugia malayi for nematode drug targets.

    Science.gov (United States)

    Kumar, Sanjay; Chaudhary, Kshitiz; Foster, Jeremy M; Novelli, Jacopo F; Zhang, Yinhua; Wang, Shiliang; Spiro, David; Ghedin, Elodie; Carlow, Clotilde K S

    2007-01-01

    We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression.

  10. Production of Brugia malayi BmSXP Recombinant Protein Expressed in Escherichia coli

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    Khoo, T. K.

    2010-01-01

    Full Text Available A rapid antibody detection test is very useful for detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. One such kit, panLF RapidTM (commercialized by Malaysian BioDiagnostic Research Sdn. Bhd. had been developed in our laboratory for the detection of all species of filarial infections. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant Brugia malayi antigens, BmR1 and BmSXP. In this study, the growth of recombinant bacteria that produce BmSXP was optimized under shake flask fermentation for high yield of the recombinant antigen. The optimizations involved selection of suitable growth medium, IPTG concentration and induction time. The medium that yielded the highest biomass as well as total protein was Terrific Broth (TB medium, which is an undefined medium. Initiation of induction of protein expression was found to be best at mid-log phase (OD600 = 1.5, with IPTG concentration of 1.0 mM, and harvest time at 9 h post-induction. This study showed that under the optimized conditions, the shake flask culture produced 4 g/L biomass (dry cell weight of recombinant Escherichia coli BmSXP/pPROEXHTa/TOP10F’, which yielded 2.42 mg/L of purified BmSXP recombinant antigen. The purified antigen was analyzed by SDS-PAGE and the antigenicity of protein was confirmed by Western blot.

  11. Potential involvement of Brugia malayi cysteine proteases in the maintenance of the endosymbiotic relationship with Wolbachia

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    Sara Lustigman

    2014-12-01

    Full Text Available Brugia malayi, a parasitic nematode that causes lymphatic filariasis, harbors endosymbiotic intracellular bacteria, Wolbachia, that are required for the development and reproduction of the worm. The essential nature of this endosymbiosis led to the development of anti-Wolbachia chemotherapeutic approaches for the treatment of human filarial infections. Our study is aimed at identifying specific proteins that play a critical role in this endosymbiotic relationship leading to the identification of potential targets in the adult worms. Filarial cysteine proteases are known to be involved in molting and embryogenesis, processes shown to also be Wolbachia dependent. Based on the observation that cysteine protease transcripts are differentially regulated in response to tetracycline treatment, we focused on defining their role in symbiosis. We observe a bimodal regulation pattern of transcripts encoding cysteine proteases when in vitro tetracycline treated worms were examined. Using tetracycline-treated infertile female worms and purified embryos we established that the first peak of the bimodal pattern corresponds to embryonic transcripts while the second takes place within the hypodermis of the adult worms. Localization studies of the native proteins corresponding to Bm-cpl-3 and Bm-cpl-6 indicate that they are present in the area surrounding Wolbachia, and, in some cases, the proteins appear localized within the bacteria. Both proteins were also found in the inner bodies of microfilariae. The possible role of these cysteine proteases during development and endosymbiosis was further characterized using RNAi. Reduction in Bm-cpl-3 and Bm-cpl-6 transcript levels was accompanied by hindered microfilarial development and release, and reduced Wolbachia DNA levels, making these enzymes strong drug target candidates.

  12. Live Brugia malayi microfilariae inhibit transendothelial migration of neutrophils and monocytes.

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    Jan-Hendrik Schroeder

    Full Text Available Lymphatic filariasis is a major tropical disease caused by the parasite Brugia malayi. Microfilariae (Mf circulate in the peripheral blood for 2-3 hours in synchronisation with maximal feeding of the mosquito vector. When absent from the peripheral blood, Mf sequester in the capillaries of the lungs. Mf are therefore in close contact with vascular endothelial cells (EC and may induce EC immune function and/or wound repair mechanisms such as angiogenesis. In this study, Mf were co-cultured with human umbilical vein EC (HUVEC or human lung microvascular EC (HLMVEC and the transendothelial migration of leukocyte subsets was analysed. In addition, the protein and/or mRNA expression of chemokine, cytokine and angiogenic mediators in endothelial cells in the presence of live microfilariae were measured by a combination of cDNA arrays, protein arrays, ELISA and fluorescence antibody tests.Surprisingly, our findings indicate that Mf presence partially blocked transendothelial migration of monocytes and neutrophils, but not lymphocytes. However, Mf exposure did not result in altered vascular EC expression of key mediators of the tethering stage of extravasation, such as ICAM-1, VCAM-1 and various chemokines. To further analyse the immunological function of vascular EC in the presence of Mf, we measured the mRNA and/or protein expression of a number of pro-inflammatory mediators. We found that expression levels of the mediators tested were predominantly unaltered upon B. malayi Mf exposure. In addition, a comparison of angiogenic mediators induced by intact Mf and Wolbachia-depleted Mf revealed that even intact Mf induce the expression of remarkably few angiogenic mediators in vascular EC. Our study suggests that live microfilariae are remarkably inert in their induction and/or activation of vascular cells in their immediate local environment. Overall, this work presents important insights into the immunological function of the vascular endothelium during

  13. Glucose and Glycogen Metabolism in Brugia malayi Is Associated with Wolbachia Symbiont Fitness.

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    Voronin, Denis; Bachu, Saheed; Shlossman, Michael; Unnasch, Thomas R; Ghedin, Elodie; Lustigman, Sara

    2016-01-01

    Wolbachia are endosymbiotic bacteria found in the majority of arthropods and filarial nematodes of medical and veterinary importance. They have evolved a wide range of symbiotic associations. In filarial nematodes that cause human lymphatic filariasis (Wuchereria bancrofti, Brugia malayi) or onchocerciasis (Onchocerca volvulus), Wolbachia are important for parasite development, reproduction and survival. The symbiotic bacteria rely in part on nutrients and energy sources provided by the host. Genomic analyses suggest that the strain of Wolbachia found in B. malayi (wBm) lacks the genes for two glycolytic enzymes--6-phosphofructokinase and pyruvate kinase--and is thus potentially unable to convert glucose into pyruvate, an important substrate for energy generation. The Wolbachia surface protein, wBm00432, is complexed to six B. malayi glycolytic enzymes, including aldolase. In this study we characterized two B. malayi aldolase isozymes and found that their expression is dependent on Wolbachia fitness and number. We confirmed by immuno-transmission electron microscopy that aldolase is associated with the Wolbachia surface. RNAi experiments suggested that aldolase-2 plays a significant role in both Wolbachia survival and embryogenesis in B. malayi. Treatment with doxycycline reduced Wolbachia fitness and increased the amount of both glucose and glycogen detected in the filarial parasite, indicating that glucose metabolism and glycogen storage in B. malayi are associated with Wolbachia fitness. This metabolic co-dependency between Wolbachia and its filarial nematode indicates that glycolysis could be a shared metabolic pathway between the bacteria and B. malayi, and thus a potential new target for anti-filarial therapy.

  14. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms.

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    Li, Ben-Wen; Rush, Amy C; Weil, Gary J

    2015-12-01

    Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of "classical" anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of V as deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects of

  15. Susceptibility of eight species members in the Anopheles hyrcanus group to nocturnally subperiodic Brugia malayi

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    Saeung Atiporn

    2013-01-01

    Full Text Available Abstract Background Filariasis, caused by Brugia malayi, is a public health problem in Thailand. Currently, at least two locations in southern Thailand are reported to be active endemic areas. Two and four Mansonia species are primary and secondary vectors, respectively, of the nocturnally subperiodic race, whereas, Coquillettidia crassipes is a vector of the diurnally subperiodic race. Although several Anopheles species have been incriminated extensively as natural and/or suspected vectors of B. malayi, little is known about vector competence between indigenous Anopheles and this filaria in Thailand. Thus, the susceptibility levels of eight species members in the Thai An. hyrcanus group to nocturnally subperiodic B. malayi are presented herein, and the two main refractory factors that affect them in different degrees of susceptibility have been elucidated. Methods Aedes togoi (a control vector, An. argyropus, An. crawfordi, An. nigerrimus, An. nitidus, An. paraliae, An. peditaeniatus, An. pursati and An. sinensis were allowed to feed artificially on blood containing B. malayi microfilariae, and dissected 14 days after feeding. To determine factors that take effect at different susceptibility levels, stain-smeared blood meals were taken from the midguts of Ae. togoi, An. peditaeniatus, An. crawfordi, An. paraliae, An. sinensis and An. nitidus immediately after feeding, and their dissected-thoraxes 4 days post blood-feedings were examined consecutively for microfilariae and L1 larvae. Results The susceptibility rates of Ae. togoi, An. peditaeniatus, An. crawfordi, An. nigerrimus, An. argyropus, An. pursati, An. sinensis, An. paraliae and An. nitidus to B. malayi were 70–95%, 70–100%, 80–85%, 50–65%, 60%, 60%, 10%, 5%, and 0%, respectively. These susceptibility rates related clearly to the degrees of normal larval development in thoracic muscles, i.e., Ae. togoi, An. peditaeniatus, An. crawfordi, An. paraliae, An. sinensis and An. nitidus

  16. Computational prediction of essential genes in an unculturable endosymbiotic bacterium, Wolbachia of Brugia malayi

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    Carlow Clotilde KS

    2009-11-01

    Full Text Available Abstract Background Wolbachia (wBm is an obligate endosymbiotic bacterium of Brugia malayi, a parasitic filarial nematode of humans and one of the causative agents of lymphatic filariasis. There is a pressing need for new drugs against filarial parasites, such as B. malayi. As wBm is required for B. malayi development and fertility, targeting wBm is a promising approach. However, the lifecycle of neither B. malayi nor wBm can be maintained in vitro. To facilitate selection of potential drug targets we computationally ranked the wBm genome based on confidence that a particular gene is essential for the survival of the bacterium. Results wBm protein sequences were aligned using BLAST to the Database of Essential Genes (DEG version 5.2, a collection of 5,260 experimentally identified essential genes in 15 bacterial strains. A confidence score, the Multiple Hit Score (MHS, was developed to predict each wBm gene's essentiality based on the top alignments to essential genes in each bacterial strain. This method was validated using a jackknife methodology to test the ability to recover known essential genes in a control genome. A second estimation of essentiality, the Gene Conservation Score (GCS, was calculated on the basis of phyletic conservation of genes across Wolbachia's parent order Rickettsiales. Clusters of orthologous genes were predicted within the 27 currently available complete genomes. Druggability of wBm proteins was predicted by alignment to a database of protein targets of known compounds. Conclusion Ranking wBm genes by either MHS or GCS predicts and prioritizes potentially essential genes. Comparison of the MHS to GCS produces quadrants representing four types of predictions: those with high confidence of essentiality by both methods (245 genes, those highly conserved across Rickettsiales (299 genes, those similar to distant essential genes (8 genes, and those with low confidence of essentiality (253 genes. These data facilitate

  17. The Wolbachia endosymbiont of Brugia malayi has an active phosphoglycerate mutase: a candidate target for anti-filarial therapies.

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    Foster, Jeremy M; Raverdy, Sylvine; Ganatra, Mehul B; Colussi, Paul A; Taron, Christopher H; Carlow, Clotilde K S

    2009-04-01

    Phosphoglycerate mutases (PGM) interconvert 2- and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. A putative cofactor-independent phosphoglycerate mutase gene (iPGM) was identified in the genome sequence of the Wolbachia endosymbiont from the filarial nematode, Brugia malayi (wBm). Since iPGM has no sequence or structural similarity to the cofactor-dependent phosphoglycerate mutase (dPGM) found in mammals, it may represent an attractive Wolbachia drug target. In the present study, wBm-iPGM cloned and expressed in Escherichia coli was mostly insoluble and inactive. However, the protein was successfully produced in the yeast Kluyveromyces lactis and the purified recombinant wBm-iPGM showed typical PGM activity. Our results provide a foundation for further development of wBm-iPGM as a promising new drug target for novel anti-filarial therapies that selectively target the endosymbiont.

  18. Treatment Follow-up of Brugia malayi Microfilaraemic and Amicrofilaraemic Individuals with Serological Evidence of Active Infection

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    Rahmah, N.

    2005-01-01

    Full Text Available Filariasis caused by Brugia malayi and Brugia timori affects ~13 million Asians. In order to ensure elimination of these infections in the context of the Global Programme for Elimination of Lymphatic Filariasis (GPELF, assays which are more sensitive than night blood examination must be employed. IgG4 assay using BmR1 recombinant antigen has been shown to be highly specific and sensitive for diagnosis of brugian filariasis. To provide further evidence of the diagnostic value of this assay, treatment follow-up study was performed on B. malayi microfilaraemic and amicrofilaraemic individuals who were positive by the BmR1-based IgG4-ELISA. Group 1 comprised 22 treated microfilaraemic individuals; group 2A comprised 13 treated amicrofilaraemic individuals and group 2B (control group comprised 16 untreated amicrofilaraemic individuals. Group 1 individuals demonstrated decline in IgG4 levels with treatment and all participants were negative by the end of the 21 months study period. Group 2A also demonstrated IgG4 decline to negativity by 21 months, with re-treatment at 12 months performed on 3 individuals. In group 2B untreated individuals, at 21 months seven participants remained IgG4 positive while nine individuals were IgG4 negative, possibly through spontaneous death of adult worms. Significant difference (p=0.008 was observed when proportions between group 2A and group 2B were compared. This study showed decline of filaria-specific IgG4 post-treatment in both microfilaria positive and microfilaria negative individuals. In addition amicrofilaraemic IgG4 positive individuals were shown to be infected as evidenced by the significant difference between treated and untreated groups of individuals. Therefore, this study strengthened the reported findings that IgG4 assay based on BmR1 recombinant antigen is a good diagnostic tool for brugian filariasis.

  19. PENENTUAN JENIS NYAMUK MansoniaSEBAGAI TERSANGKA VEKTOR FILARIASIS Brugia malayi DAN HEWAN ZOONOSIS DI KABUPATEN MUARO JAMBI

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    Santoso Santoso

    2015-01-01

    Full Text Available AbstrakFilariasis merupakan penyakit yang tidak mudah menular. Filariasis adalah penyakit yang ditularkan oleh nyamuk sebagai vector. Jenis nyamuk yang dapat berperan sebagai vector filariasis dipengaruhi oleh jenis cacing penyebab filaria. Brugia spp. umumnya ditularkan oleh nyamuk Mansonia spp dan Anopheles spp. Vektor dan hewan zoonosis merupakan salah satu factor yang dapat perlu mendapat perhatian dalam pengendalian filariasis. Penelitian terhadap vector dan hewan zoonosis telah dilakukan di Kabupaten Muaro Jambi untuk mengidentifikasi bionomik vektor dan kemungkinan adanya hewan zoonosis yang berperan sebagai penular filariasis. Desain penelitian adalah observasi, yaitu dengan melakukan penangkapan nyamuk dan pemeriksaan darah terhadap kucing. Jumlah kucing yang diperiksa sebanyak 18 ekor. Kucing yang positif microfilaria sebanyak 1 ekor. Jumlah nyamuk Mansonia spp. tertangkap sebanyak 1,167 ekor yang terdiri dari 6 species. Spesies nyamuk tertangkap paling banyak adalah Mansonia uniformis sebanyak 1.010 ekor dengan angka kekerapan 1,0. Berdasarkan hasil tersebut, maka diperlukan peran serta masyarakat untuk mengurangi kepadatan nyamuk dengan membersihkan genangan air dan mencegah gigitan nyamuk. Selain itu diperlukan juga penanganan terhadap hewan yang bertindak sebagai zoonosis dengan memberikan pengobatan terhadap kucing agar tidak menjadi sumber infeksi.Keywords : filariasis, Mansonia, vektor, zoonosis, Muaro Jambi.AbstractFilariasisis noteasily transmitted diseases. Filariasisis transmitted by mosquito vectors. Various types of mosquitoes can act as vectors of filariasis, depending on the type of microfilaria. Brugia spp. are generally transmitted by Mansonia spp and Anopheles spp. Vector and zoonotic animal are the factors that can transmit filariasis and need to have attention for controlling filariasis. Research on vector and zoonotic had been done in Muaro Jambi to determine bionomic vector and the possibility of animals can

  20. Comparative analysis of ITS1 nucleotide sequence reveals distinct genetic difference between Brugia malayi from Northeast Borneo and Thailand.

    Science.gov (United States)

    Fong, Mun-Yik; Noordin, Rahmah; Lau, Yee-Ling; Cheong, Fei-Wen; Yunus, Muhammad Hafiznur; Idris, Zulkarnain Md

    2013-01-01

    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.

  1. Filaria-induced immune evasion: suppression by the infective stage of Brugia malayi at the earliest host-parasite interface.

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    Semnani, Roshanak Tolouei; Law, Melissa; Kubofcik, Joseph; Nutman, Thomas B

    2004-05-15

    To assess the physiologic interactions between the infective stage of Brugia malayi--one of the extracellular parasites responsible for lymphatic filariasis in humans--and the APC with which they come in contact during their development and routes of travel, we have investigated the interaction between the infective stage (L3) of B. malayi and human Langerhans cells (LC) in the skin. Our data indicate that live L3 result in increased migration of LC from the epidermis without affecting the viability of these cells and up-regulation of the IL-18 cytokine involved in LC migration. Live L3 also result in down-regulation of MHC class I and II on the LC cell surface. Additionally, microarray data indicate that live L3 significantly down-regulated expression of IL-8 as well as of multiple genes involved in Ag presentation, reducing the capacity of LC to induce CD4(+) T cells in allogeneic MLR, and thus resulting in a decreased ability of LC to promote CD4(+) T cell proliferation and production of IFN-gamma and IL-10. These data suggest that L3 exert a down-regulatory response in epidermal LC that leads to a diminished capacity of these cells to activate CD4(+) T cells.

  2. Immunological evaluation of an rsmD-like rRNA methyltransferase from Wolbachia endosymbiont of Brugia malayi.

    Science.gov (United States)

    Rana, Ajay Kumar; Kushwaha, Susheela; Singh, Prashant Kumar; Misra-Bhattacharya, Shailja

    2016-02-01

    Wolbachia is a wonderful anti-filarial target with many of its enzymes and surface proteins (WSPs) representing potential drug targets and vaccine candidates. Here we report on the immunologic response of a drug target, rsmD-like rRNA methyltransferase from Wolbachia endosymbiont of Brugia malayi. The recombinant protein generated both humoral and cell-mediated response in BALB/c mice but compromised its immunity. The humoral response was transient and endured barely for six months in mice with or without B. Malayi challenge. In splenocytes of mice, the key humoral immunity mediating cytokine IL4 was lowered (IL4↓) while IFNγ, the major cytokine mediating cellular immunity was decreased along with upregulation of IL10 cytokine (IFNγ↓, IL10↑). The finding here indicates that the enzyme has low immunogenicity and triggers lowering of cytokine level in BALB/c mice. Interestingly the overall immune profile can be summed up with equivalent response generated by WSP or whole Wolbachia.

  3. Detection of filaria-specific IgG4 antibodies and filarial DNA, for the screening of blood spots for Brugia timori.

    Science.gov (United States)

    Fischer, P; Bonow, I; Supali, T; Rückert, P; Rahmah, N

    2005-01-01

    The establishment of simple, sensitive and specific tools for the diagnosis of brugian lymphatic filariasis is a prerequisite for a successful intervention to control the disease. In the simple and rapid Brugia Rapid (BR) test, an immunochromatographic dipstick is used to detect IgG(4) antibodies that are reactive with a recombinant Brugia malayi antigen. When sera from 109 individuals with Brugia microfilaraemias (12 with B. malayi and 97 with B. timori) were investigated using the BR test, all were found positive. In contrast, all of the 150 sera from individuals with Onchocerca volvulus or Mansonella infections investigated were found negative in BR tests. Some unwelcome cross-reactions were observed, however, with sera from individuals infected with Wuchereria bancrofti (three of 12 test-positive) and Dirofilaria (one of nine test-positive). In an attempt to facilitate sample collection and detect any cross-reactions, the BR dipstick was used to screen blood spots, that had been allowed to dry on filter paper, for B. timori microfilariae, before the dipstick-positive samples were tested with a PCR-based assay. Of the 66 individuals so tested, 37 (56%) were found positive by the BR test used on dry blood spots and eight (22%) by the filtration of fresh blood samples. Only nine of the 37 dipstick-positive samples were found PCR-positive. The combined use of BR tests and PCR-based assays, for testing blood spots in areas where brugian filariasis is endemic, appears to be a promising method not only for post-treatment monitoring but also for the certification activities planned within the framework of the Global Programme to Eliminate Lymphatic Filariasis.

  4. A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans.

    OpenAIRE

    Takacs, A M; Denker, J A; Perrine, K G; Maroney, P A; Nilsen, T W

    1988-01-01

    The mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt se...

  5. Diversity in parasitic nematode genomes: the microRNAs of Brugia pahangi and Haemonchus contortus are largely novel

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    Winter Alan D

    2012-01-01

    Full Text Available Abstract Background MicroRNAs (miRNAs play key roles in regulating post-transcriptional gene expression and are essential for development in the free-living nematode Caenorhabditis elegans and in higher organisms. Whether microRNAs are involved in regulating developmental programs of parasitic nematodes is currently unknown. Here we describe the the miRNA repertoire of two important parasitic nematodes as an essential first step in addressing this question. Results The small RNAs from larval and adult stages of two parasitic species, Brugia pahangi and Haemonchus contortus, were identified using deep-sequencing and bioinformatic approaches. Comparative analysis to known miRNA sequences reveals that the majority of these miRNAs are novel. Some novel miRNAs are abundantly expressed and display developmental regulation, suggesting important functional roles. Despite the lack of conservation in the miRNA repertoire, genomic positioning of certain miRNAs within or close to specific coding genes is remarkably conserved across diverse species, indicating selection for these associations. Endogenous small-interfering RNAs and Piwi-interacting (piRNAs, which regulate gene and transposon expression, were also identified. piRNAs are expressed in adult stage H. contortus, supporting a conserved role in germline maintenance in some parasitic nematodes. Conclusions This in-depth comparative analysis of nematode miRNAs reveals the high level of divergence across species and identifies novel sequences potentially involved in development. Expression of novel miRNAs may reflect adaptations to different environments and lifestyles. Our findings provide a detailed foundation for further study of the evolution and function of miRNAs within nematodes and for identifying potential targets for intervention.

  6. Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi

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    Landmann Frédéric

    2012-01-01

    Full Text Available Abstract Background RNA interference (RNAi is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans. Results We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week. Conclusions We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis.

  7. Brugia malayi microfilariae induce a regulatory monocyte/macrophage phenotype that suppresses innate and adaptive immune responses.

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    Noëlle Louise O'Regan

    2014-10-01

    Full Text Available Monocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive.To determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses.Monocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4+ T cell proliferation and cytokine production (IFN-γ, IL-13 and IL-10. IFN-γ responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner.Conclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression of PD-L1 and IL-10. This phenotype is directly reflected in monocytes from filarial patients with asymptomatic infection but not patients with pathology or endemic normals. We suggest that suppression of T cell functions typically seen in lymphatic filariasis is caused by microfilaria-modulated monocytes in an IL

  8. Brugia malayi Microfilariae Induce a Regulatory Monocyte/Macrophage Phenotype That Suppresses Innate and Adaptive Immune Responses

    Science.gov (United States)

    Venugopal, Gopinath; Rao, Gopala B.; Lucius, Richard; Srikantam, Aparna; Hartmann, Susanne

    2014-01-01

    Background Monocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive. Aim To determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses. Methodology and principal findings Monocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf) lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4+ T cell proliferation and cytokine production (IFN-γ, IL-13 and IL-10). IFN-γ responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner. Conclusions and significance Conclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression of PD-L1 and IL-10. This phenotype is directly reflected in monocytes from filarial patients with asymptomatic infection but not patients with pathology or endemic normals. We suggest that suppression of T cell functions typically seen in lymphatic

  9. Brugia malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-specific proteomic profiling.

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    Sasisekhar Bennuru

    Full Text Available Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf, L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs in the available databases. Moreover, this analysis was able to confirm the presence of 274 "hypothetical" proteins inferred from gene prediction algorithms applied to the B. malayi (Bm genome. Not surprisingly, the majority (160/274 of these "hypothetical" proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase, MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host-parasite interaction.

  10. Structure of the trehalose-6-phosphate phosphatase from Brugia malayi reveals key design principles for anthelmintic drugs.

    Science.gov (United States)

    Farelli, Jeremiah D; Galvin, Brendan D; Li, Zhiru; Liu, Chunliang; Aono, Miyuki; Garland, Megan; Hallett, Olivia E; Causey, Thomas B; Ali-Reynolds, Alana; Saltzberg, Daniel J; Carlow, Clotilde K S; Dunaway-Mariano, Debra; Allen, Karen N

    2014-07-01

    Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.

  11. Molecular characterization of NAD+-dependent DNA ligase from Wolbachia endosymbiont of lymphatic filarial parasite Brugia malayi.

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    Nidhi Shrivastava

    Full Text Available The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies.

  12. Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference.

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    Louise Ford

    Full Text Available BACKGROUND: Cathepsin-like enzymes have been identified as potential targets for drug or vaccine development in many parasites, as their functions appear to be essential in a variety of important biological processes within the host, such as molting, cuticle remodeling, embryogenesis, feeding and immune evasion. Functional analysis of Caenorhabditis elegans cathepsin L (Ce-cpl-1 and cathepsin Z (Ce-cpz-1 has established that both genes are required for early embryogenesis, with Ce-cpl-1 having a role in regulating in part the processing of yolk proteins. Ce-cpz-1 also has an important role during molting. METHODS AND FINDINGS: RNA interference assays have allowed us to verify whether the functions of the orthologous filarial genes in Brugia malayi adult female worms are similar. Treatment of B. malayi adult female worms with Bm-cpl-1, Bm-cpl-5, which belong to group Ia of the filarial cpl gene family, or Bm-cpz-1 dsRNA resulted in decreased numbers of secreted microfilariae in vitro. In addition, analysis of the intrauterine progeny of the Bm-cpl-5 or Bm-cpl Pro dsRNA- and siRNA-treated worms revealed a clear disruption in the process of embryogenesis resulting in structural abnormalities in embryos and a varied differential development of embryonic stages. CONCLUSIONS: Our studies suggest that these filarial cathepsin-like cysteine proteases are likely to be functional orthologs of the C. elegans genes. This functional conservation may thus allow for a more thorough investigation of their distinct functions and their development as potential drug targets.

  13. A deep sequencing approach to comparatively analyze the transcriptome of lifecycle stages of the filarial worm, Brugia malayi.

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    Young-Jun Choi

    2011-12-01

    Full Text Available BACKGROUND: Developing intervention strategies for the control of parasitic nematodes continues to be a significant challenge. Genomic and post-genomic approaches play an increasingly important role for providing fundamental molecular information about these parasites, thus enhancing basic as well as translational research. Here we report a comprehensive genome-wide survey of the developmental transcriptome of the human filarial parasite Brugia malayi. METHODOLOGY/PRINCIPAL FINDINGS: Using deep sequencing, we profiled the transcriptome of eggs and embryos, immature (≤3 days of age and mature microfilariae (MF, third- and fourth-stage larvae (L3 and L4, and adult male and female worms. Comparative analysis across these stages provided a detailed overview of the molecular repertoires that define and differentiate distinct lifecycle stages of the parasite. Genome-wide assessment of the overall transcriptional variability indicated that the cuticle collagen family and those implicated in molting exhibit noticeably dynamic stage-dependent patterns. Of particular interest was the identification of genes displaying sex-biased or germline-enriched profiles due to their potential involvement in reproductive processes. The study also revealed discrete transcriptional changes during larval development, namely those accompanying the maturation of MF and the L3 to L4 transition that are vital in establishing successful infection in mosquito vectors and vertebrate hosts, respectively. CONCLUSIONS/SIGNIFICANCE: Characterization of the transcriptional program of the parasite's lifecycle is an important step toward understanding the developmental processes required for the infectious cycle. We find that the transcriptional program has a number of stage-specific pathways activated during worm development. In addition to advancing our understanding of transcriptome dynamics, these data will aid in the study of genome structure and organization by facilitating

  14. Development of an in vivo RNAi protocol to investigate gene function in the filarial nematode, Brugia malayi.

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    Chuanzhe Song

    Full Text Available Our ability to control diseases caused by parasitic nematodes is constrained by a limited portfolio of effective drugs and a paucity of robust tools to investigate parasitic nematode biology. RNA interference (RNAi is a reverse-genetics tool with great potential to identify novel drug targets and interrogate parasite gene function, but present RNAi protocols for parasitic nematodes, which remove the parasite from the host and execute RNAi in vitro, are unreliable and inconsistent. We have established an alternative in vivo RNAi protocol targeting the filarial nematode Brugia malayi as it develops in an intermediate host, the mosquito Aedes aegypti. Injection of worm-derived short interfering RNA (siRNA and double stranded RNA (dsRNA into parasitized mosquitoes elicits suppression of B. malayi target gene transcript abundance in a concentration-dependent fashion. The suppression of this gene, a cathepsin L-like cysteine protease (Bm-cpl-1 is specific and profound, both injection of siRNA and dsRNA reduce transcript abundance by 83%. In vivo Bm-cpl-1 suppression results in multiple aberrant phenotypes; worm motility is inhibited by up to 69% and parasites exhibit slow-moving, kinked and partial-paralysis postures. Bm-cpl-1 suppression also retards worm growth by 48%. Bm-cpl-1 suppression ultimately prevents parasite development within the mosquito and effectively abolishes transmission potential because parasites do not migrate to the head and proboscis. Finally, Bm-cpl-1 suppression decreases parasite burden and increases mosquito survival. This is the first demonstration of in vivo RNAi in animal parasitic nematodes and results indicate this protocol is more effective than existing in vitro RNAi methods. The potential of this new protocol to investigate parasitic nematode biology and to identify and validate novel anthelmintic drug targets is discussed.

  15. Structure of the trehalose-6-phosphate phosphatase from Brugia malayi reveals key design principles for anthelmintic drugs.

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    Jeremiah D Farelli

    2014-07-01

    Full Text Available Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.

  16. Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

    Science.gov (United States)

    Thanchomnang, Tongjit; Intapan, Pewpan M; Tantrawatpan, Chairat; Lulitanond, Viraphong; Chungpivat, Sudchit; Taweethavonsawat, Piyanan; Kaewkong, Worasak; Sanpool, Oranuch; Janwan, Penchom; Choochote, Wej; Maleewong, Wanchai

    2013-12-01

    A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

  17. The heme biosynthetic pathway of the obligate Wolbachia endosymbiont of Brugia malayi as a potential anti-filarial drug target.

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    Bo Wu

    Full Text Available BACKGROUND: Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole can interrupt transmission predominantly by killing microfilariae (mf larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs. METHODS AND FINDINGS: Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP, which targets ferrochelatase (FC, the last step. Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a approximately 600-fold difference in drug sensitivities to succinyl acetone (SA between

  18. Characterization of transcription factors that regulate the type IV secretion system and riboflavin biosynthesis in Wolbachia of Brugia malayi.

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    Zhiru Li

    Full Text Available The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium Wolbachia (wBm that is required for parasite survival. Consequently, targeting wBm is a promising approach for anti-filarial drug development. The Type IV secretion system (T4SS plays an important role in bacteria-host interactions and is under stringent regulation by transcription factors. In wBm, most T4SS genes are contained in two operons. We show the wBm is active since the essential assembly factor virB8-1, is transcribed in adult worms and larval stages, and VirB8-1 is present in parasite lysates. We also identify two transcription factors (wBmxR1 and wBmxR2 that bind to the promoter region of several genes of the T4SS. Gel shift assays show binding of wBmxR1 to regions upstream of the virB9-2 and wBmxR2 genes, whereas wBmxR2 binds to virB4-2 and wBmxR1 promoter regions. Interestingly, both transcription factors bind to the promoter of the ribA gene that precedes virB8-1, the first gene in operon 1 of the wBm T4SS. RT-PCR reveals ribA and virB8-1 genes are co-transcribed as one operon, indicating the ribA gene and T4SS operon 1 are co-regulated by both wBmxR1 and wBmxR2. RibA encodes a bi-functional enzyme that catalyzes two essential steps in riboflavin (Vitamin B2 biosynthesis. Importantly, the riboflavin pathway is absent in B. malayi. We demonstrate the pathway is functional in wBm, and observe vitamin B2 supplementation partially rescues filarial parasites treated with doxycycline, indicating Wolbachia may supply the essential vitamin to its worm host. This is the first characterization of a transcription factor(s from wBm and first report of co-regulation of genes of the T4SS and riboflavin biosynthesis pathway. In addition, our results demonstrate a requirement of vitamin B2 for worm health and fertility, and imply a nutritional role of the symbiont for the filarial parasite host.

  19. Characterization of transcription factors that regulate the type IV secretion system and riboflavin biosynthesis in Wolbachia of Brugia malayi.

    Science.gov (United States)

    Li, Zhiru; Carlow, Clotilde K S

    2012-01-01

    The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium Wolbachia (wBm) that is required for parasite survival. Consequently, targeting wBm is a promising approach for anti-filarial drug development. The Type IV secretion system (T4SS) plays an important role in bacteria-host interactions and is under stringent regulation by transcription factors. In wBm, most T4SS genes are contained in two operons. We show the wBm is active since the essential assembly factor virB8-1, is transcribed in adult worms and larval stages, and VirB8-1 is present in parasite lysates. We also identify two transcription factors (wBmxR1 and wBmxR2) that bind to the promoter region of several genes of the T4SS. Gel shift assays show binding of wBmxR1 to regions upstream of the virB9-2 and wBmxR2 genes, whereas wBmxR2 binds to virB4-2 and wBmxR1 promoter regions. Interestingly, both transcription factors bind to the promoter of the ribA gene that precedes virB8-1, the first gene in operon 1 of the wBm T4SS. RT-PCR reveals ribA and virB8-1 genes are co-transcribed as one operon, indicating the ribA gene and T4SS operon 1 are co-regulated by both wBmxR1 and wBmxR2. RibA encodes a bi-functional enzyme that catalyzes two essential steps in riboflavin (Vitamin B2) biosynthesis. Importantly, the riboflavin pathway is absent in B. malayi. We demonstrate the pathway is functional in wBm, and observe vitamin B2 supplementation partially rescues filarial parasites treated with doxycycline, indicating Wolbachia may supply the essential vitamin to its worm host. This is the first characterization of a transcription factor(s) from wBm and first report of co-regulation of genes of the T4SS and riboflavin biosynthesis pathway. In addition, our results demonstrate a requirement of vitamin B2 for worm health and fertility, and imply a nutritional role of the symbiont for the filarial parasite host.

  20. Designing, synthesis of selective and high-affinity chalcone-benzothiazole hybrids as Brugia malayi thymidylate kinase inhibitors: In vitro validation and docking studies.

    Science.gov (United States)

    Sashidhara, Koneni V; Avula, Srinivasa Rao; Doharey, Pawan Kumar; Singh, L Ravithej; Balaramnavar, Vishal M; Gupta, Jyoti; Misra-Bhattacharya, Shailja; Rathaur, Sushma; Saxena, Anil K; Saxena, Jitendra Kumar

    2015-10-20

    In our continuing search for safe and efficacious antifilarials, a series of novel chalcone-benzothiazole hybrids have been synthesized and evaluated for their Brugia malayi thymidylate kinase (BmTMK) enzyme inhibition activity. Their selectivity towards BmTMK was studied and compared to the human TMK (HsTMK) by an in silico method. Out of seventeen derivatives, compounds 34 and 42 showed higher interactions with the BmTMK active site. MolDock docking model revealed the interactions of these two derivatives and the results corroborated well with their in vitro antifilarial activities. Our studies suggest that these hybrids are selective towards the BmTMK enzyme and may serve as potential therapeutic agents against filariasis.

  1. Low positive predictive value of anti-Brugia malayi IgG and IgG4 serology for the diagnosis of Wuchereria bancrofti.

    Science.gov (United States)

    Chanteau, S; Glaziou, P; Moulia-Pelat, J P; Plichart, C; Luquiaud, P; Cartel, J L

    1994-01-01

    Enzyme-linked immunosorbent assays (ELISAs) for anti-Brugia malayi immunoglobulin (Ig) G and IgG4 were evaluated on sera from 1561 subjects in French Polynesia for the serodiagnosis of Wuchereria bancrofti filariasis, compared with the test for Onchocerca gibsoni circulating antigen (Og4C3) as a 'gold standard'. The sensitivity of the ELISA-IgG and ELISA-IgG4 assays was 90.8% and 94.5%, and the specificity was 45.9% and 50.7%. The positive predictive values were 41% and 45% respectively for an antigen prevalence rate of 30%. Thus antibody prevalences exceeded by two-fold the antigen prevalence, which itself exceeded by two-fold the prevalence of microfilaraemia.

  2. Experimental chemotherapy of filariasis: comparative evaluation of the efficacy of filaricidal compounds in Mastomys coucha infected with Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi and B. pahangi.

    Science.gov (United States)

    Zahner, H; Schares, G

    1993-01-01

    Eleven types/classes of compound with antifilarial activity were comparatively evaluated in Mastomys coucha infected with Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi or B. pahangi. The paper deals with the efficacy of (i) predominantly microfilaricidal compounds [diethylcarbamazine, levamisole, avermectins (ivermectin, milbemycin), nitrofurans (nitrofurantoin, hydroxymethylnitrofurantoin, nifurtimox, furazolidone, furapyrimidone), organophosphorals (metrifonate, haloxon), and aminophenyl-amidines], (ii) predominantly macrofilaricidal compounds [suramin, benzimidazoles (flubendazole, mebendazole, oxfendazole, ciclobendazole, albendazole, cambendazole, fenbendazole), and arsenicals (thiacetarsamide, Mel PH, R7/45)], and (iii) micro- and macrofilaricidal compounds [benzazole derivatives (CGP 20376 and other benzothiazoles) and nitrophenylamines (amoscanate, CGP 6140)]. Minimum effective doses against microfilariae and minimum curative doses against adult filariae as well as detailed data on dose-efficacy relationships are reported for the various drugs. The results obtained in M. coucha are compared with those published for other experimental in vivo filarial systems, thus attempting to describe a general status of in vivo antifilarial activity of the compounds.

  3. Suppression of Brugia malayi (sub-periodic larval development in Aedes aegypti (Liverpool strain fed on blood of animals immunized with microfilariae

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    K Athisaya Mary

    2005-07-01

    Full Text Available Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic, in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain. In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3. Fewer number of parasites developed to first stage (L1 and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.

  4. Cofactor Independent Phosphoglycerate Mutase of Brugia malayi Induces a Mixed Th1/Th2 Type Immune Response and Inhibits Larval Development in the Host

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    Prashant K. Singh

    2014-01-01

    Full Text Available Lymphatic filariasis is a major debilitating disease, endemic in 72 countries putting more than 1.39 billion people at risk and 120 million are already infected. Despite the significant progress in chemotherapeutic advancements, there is still need for other measures like development of an effective vaccine or discovery of novel drug targets. In this study, structural and immunological characterization of independent phosphoglycerate mutase of filarial parasite Brugia malayi was carried out. Protein was found to be expressed in all major parasite life stages and as an excretory secretory product of adult parasites. Bm-iPGM also reacted to all the categories of human bancroftian patient’s sera including endemic normals. In vivo immunological behaviour of protein was determined in immunized BALB/c mice followed by prophylactic analysis in BALB/c mice and Mastomys coucha. Immunization with Bm-iPGM led to generation of a mixed Th1/Th2 type immune response offering 58.2% protection against larval challenge in BALB/c and 65–68% protection in M. coucha. In vitro studies confirmed participation of anti-Bm-iPGM antibodies in killing of B. malayi infective larvae and microfilariae through ADCC mechanism. The present findings reveal potential immunoprotective nature of Bm-iPGM advocating its worth as an antifilarial vaccine candidate.

  5. Bm-CPI-2, a cystatin from Brugia malayi nematode parasites, differs from Caenorhabditis elegans cystatins in a specific site mediating inhibition of the antigen-processing enzyme AEP.

    Science.gov (United States)

    Murray, Janice; Manoury, Bénédicte; Balic, Adam; Watts, Colin; Maizels, Rick M

    2005-02-01

    The filarial parasite Brugia malayi survives for many years in the human lymphatic system. One immune evasion mechanism employed by Brugia is thought to be the release of cysteine protease inhibitors (cystatins), and we have previously shown that the recombinant cystatin Bm-CPI-2 interferes with protease-dependent antigen processing in the MHC class II antigen presentation pathway. Analogy with vertebrate cystatins suggested that Bm-CPI-2 is bi-functional, with one face of the protein blocking papain-like proteases, and the other able to inhibit legumains such as asparaginyl endopeptidase (AEP). Site-directed mutagenesis was carried out on Bm-CPI-2 at Asn-77, the residue on which AEP inhibition is dependent in vertebrate homologues. Two mutations at this site (to Asp and Lys) showed 10-fold diminished and ablated activity respectively, in assays of AEP inhibition, while blocking of papain-like proteases was reduced by only a small degree. Comparison of the B. malayi cystatins with two homologues encoded by the free-living model organism, Caenorhabditis elegans, suggested that while the papain site may be intact, the AEP site would not be functional. This supposition was tested with recombinant C. elegans proteins, Ce-CPI-1 (K08B4.6) and Ce-CPI-2 (R01B10.1), both of which block cathepsins and neither of which possess the ability to block AEP. Thus, Brugia CPI-2 may have convergently evolved to inhibit an enzyme important only in the mammalian environment.

  6. A recombinant plasmid of composite cysteine proteinase inhibitor/glyceraldehyde-3-phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host

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    Z Fang

    2016-01-01

    Full Text Available Objectives: Both cysteine proteinase inhibitors (CPIs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses. Materials and Methods: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI of T-lymphocyte proliferation and the levels of interferon-gamma (INF-g and interleukin-4 ( IL-4 in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays. Results: The pcDNA3.1(+-BmCPI/BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05. The levels of INF-g and IL-4 of pcDNA3.1(+-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05. The level of INF-g of pcDNA3.1(+-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+-BmCPI/CpG group (P < 0.05. Conclusions: We conclude that the recombinant plasmid pcDNA3.1(+-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

  7. Cloning, expression and characterization of UDP-N-acetylglucosamine enolpyruvyl transferase (MurA from Wolbachia endosymbiont of human lymphatic filarial parasite Brugia malayi.

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    Mohd Shahab

    Full Text Available Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (Km for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds.

  8. Inflammatory mediator release byBrugia malayi from macrophages of susceptible hostMastomys coucha andTHP-1 andRAW 264.7 cell lines

    Institute of Scientific and Technical Information of China (English)

    Shiv Kumar Verma; Vikas Kushwaha; Vijaya Dubey; Kirti Saxena; Aakanksha Sharma; Puvvada Kalpana Murthy

    2011-01-01

    Objective:To investigate which life stage of the parasite has the ability to stimulate release of pro- or anti-inflammatory mediators from macrophages.Methods: The human macrophage/monocyte cell lineTHP-1, the mouse macrophage cell lineRAW 264.7 and naive peritoneal macrophages(PM)from the rodent hostMastomys coucha (M. coucha)were incubated at37 ℃in 5% CO2atmosphere with extracts of microfilariae(Mf), third stage infective larvae(L3) and adult worms (Ad)ofBrugia malayi. After48 hr post exposure,IL-1β, IL-6, TNF-α, IL-10 and nitric oxide (NO) in cell-free supernatants were estimated.Results: Extracts of all the life stages of the parasite were capable of stimulating pro-(IL-1β, IL-6 andTNF-α) and anti-inflammatory (IL-10)cytokines in both the cell lines and peritoneal macrophages ofM. coucha. Mf was the strongest stimulator of pro-inflammatory cytokines followed by L3 and Ad; however, Ad was a strong stimulator ofIL-10 release. Mf was found to have potential to modulateLPS-inducedNO release inRAW cells. Ad-inducedNO release was concentration dependent with maximum at 20 μg/mL in bothRAW andPMs.Conclusions:The results show that parasites at all life stages were capable of stimulating pro- (IL-1β, IL-6 and TNF-α) and anti-inflammatory(IL-10) cytokines andNO release from macrophages of susceptible hostM. coucha, human and mouse macrophage cell lines.Mf can suppress theLPS-inducedNO release inRAW cells. The findings also show that the two cell lines may provide a convenientin vitro system for assaying parasite-induced inflammatory mediator release.

  9. Exome and Transcriptome Sequencing of Aedes aegypti Identifies a Locus That Confers Resistance to Brugia malayi and Alters the Immune Response

    KAUST Repository

    Juneja, Punita

    2015-03-27

    Many mosquito species are naturally polymorphic for their abilities to transmit parasites, a feature which is of great interest for controlling vector-borne disease. Aedes aegypti, the primary vector of dengue and yellow fever and a laboratory model for studying lymphatic filariasis, is genetically variable for its capacity to harbor the filarial nematode Brugia malayi. The genome of Ae. aegypti is large and repetitive, making genome resequencing difficult and expensive. We designed exome captures to target protein-coding regions of the genome, and used association mapping in a wild Kenyan population to identify a single, dominant, sex-linked locus underlying resistance. This falls in a region of the genome where a resistance locus was previously mapped in a line established in 1936, suggesting that this polymorphism has been maintained in the wild for the at least 80 years. We then crossed resistant and susceptible mosquitoes to place both alleles of the gene into a common genetic background, and used RNA-seq to measure the effect of this locus on gene expression. We found evidence for Toll, IMD, and JAK-STAT pathway activity in response to early stages of B. malayi infection when the parasites are beginning to die in the resistant genotype. We also found that resistant mosquitoes express anti-microbial peptides at the time of parasite-killing, and that this expression is suppressed in susceptible mosquitoes. Together, we have found that a single resistance locus leads to a higher immune response in resistant mosquitoes, and we identify genes in this region that may be responsible for this trait.

  10. Homology modeling of NAD+-dependent DNA ligase of the Wolbachia endosymbiont of Brugia malayi and its drug target potential using dispiro-cycloalkanones.

    Science.gov (United States)

    Shrivastava, Nidhi; Nag, Jeetendra K; Pandey, Jyoti; Tripathi, Rama Pati; Shah, Priyanka; Siddiqi, Mohammad Imran; Misra-Bhattacharya, Shailja

    2015-07-01

    Lymphatic filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility, and viability and thus has great promise as a novel approach for treating filarial diseases. NAD(+)-dependent DNA ligase is an essential enzyme of DNA replication, repair, and recombination. Therefore, in the present study, the antifilarial drug target potential of the NAD(+)-dependent DNA ligase of the Wolbachia symbiont of Brugia malayi (wBm-LigA) was investigated using dispiro-cycloalkanone compounds. Dispiro-cycloalkanone specifically inhibited the nick-closing and cohesive-end ligation activities of the enzyme without inhibiting human or T4 DNA ligase. The mode of inhibition was competitive with the NAD(+) cofactor. Docking studies also revealed the interaction of these compounds with the active site of the target enzyme. The adverse effects of these inhibitors were observed on adult and microfilarial stages of B. malayi in vitro, and the most active compounds were further monitored in vivo in jirds and mastomys rodent models. Compounds 1, 2, and 5 had severe adverse effects in vitro on the motility of both adult worms and microfilariae at low concentrations. Compound 2 was the best inhibitor, with the lowest 50% inhibitory concentration (IC50) (1.02 μM), followed by compound 5 (IC50, 2.3 μM) and compound 1 (IC50, 2.9 μM). These compounds also exhibited the same adverse effect on adult worms and microfilariae in vivo (P < 0.05). These compounds also tremendously reduced the wolbachial load, as evident by quantitative real-time PCR (P < 0.05). wBm-LigA thus shows great promise as an antifilarial drug target, and dispiro-cycloalkanone compounds show great promise as antifilarial lead candidates.

  11. Wolbachia endosymbiont of Brugia malayi elicits a T helper type 17-mediated pro-inflammatory immune response through Wolbachia surface protein.

    Science.gov (United States)

    Pathak, Manisha; Verma, Meenakshi; Srivastava, Mrigank; Misra-Bhattacharya, Shailja

    2015-02-01

    Wolbachia is an endosymbiotic bacterium of the filarial nematode Brugia malayi. The symbiotic relationship between Wolbachia and its filarial host is dependent on interactions between the proteins of both organisms. However, little is known about Wolbachia proteins that are involved in the inflammatory pathology of the host during lymphatic filariasis. In the present study, we cloned, expressed and purified Wolbachia surface protein (r-wsp) from Wolbachia and administered it to mice, either alone or in combination with infective larvae of B. malayi (Bm-L3) and monitored the developing immune response in infected animals. Our results show that spleens and mesenteric lymph nodes of mice immunized with either r-wsp or infected with Bm-L3 show increased percentages of CD4(+) T helper type 17 (Th17) cells and Th1 cytokines like interferon-γ and interleukin-2 (IL-2) along with decreased percentages of regulatory T cells, Th2 cytokines like IL-4 and IL-10 and transforming growth factor β (TGF-β) levels in culture supernatants of splenocytes. These observations were stronger in mice immunized with r-wsp alone. Interestingly, when mice were first immunized with r-wsp and subsequently infected with Bm-L3, percentages of CD4(+) Th17 cells and Th1 cytokines increased even further while that of regulatory T cells, Th2 cytokines and TGF-β levels decreased. These results for the first time show that r-wsp acts synergistically with Bm-L3 in promoting a pro-inflammatory response by increasing Th17 cells and at the same time diminishes host immunological tolerance by decreasing regulatory T cells and TGF-β secretion.

  12. Immunogenicity and Protective Efficacy of Brugia malayi Heavy Chain Myosin as Homologous DNA, Protein and Heterologous DNA/Protein Prime Boost Vaccine in Rodent Model.

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    Jyoti Gupta

    Full Text Available We earlier demonstrated the immunoprophylactic efficacy of recombinant heavy chain myosin (Bm-Myo of Brugia malayi (B. malayi in rodent models. In the current study, further attempts have been made to improve this efficacy by employing alternate approaches such as homologous DNA (pcD-Myo and heterologous DNA/protein prime boost (pcD-Myo+Bm-Myo in BALB/c mouse model. The gene bm-myo was cloned in a mammalian expression vector pcDNA 3.1(+ and protein expression was confirmed in mammalian Vero cell line. A significant degree of protection (79.2%±2.32 against L3 challenge in pcD-Myo+Bm-Myo immunized group was observed which was much higher than that exerted by Bm-Myo (66.6%±2.23 and pcD-Myo (41.6%±2.45. In the heterologous immunized group, the percentage of peritoneal leukocytes such as macrophages, neutrophils, B cells and T cells marginally increased and their population augmented further significantly following L3 challenge. pcD-Myo+Bm-Myo immunization elicited robust cellular and humoral immune responses as compared to pcD-Myo and Bm-Myo groups as evidenced by an increased accumulation of CD4+, CD8+ T cells and CD19+ B cells in the mouse spleen and activation of peritoneal macrophages. Though immunized animals produced antigen-specific IgG antibodies and isotypes, sera of mice receiving pcD-Myo+Bm-Myo or Bm-Myo developed much higher antibody levels than other groups and there was profound antibody-dependent cellular adhesion and cytotoxicity (ADCC to B. malayi infective larvae (L3. pcD-Myo+Bm-Myo as well as Bm-Myo mice generated a mixed T helper cell phenotype as evidenced by the production of both pro-inflammatory (IL-2, IFN-γ and anti-inflammatory (IL-4, IL-10 cytokines. Mice receiving pcD-Myo on contrary displayed a polarized pro-inflammatory immune response. The findings suggest that the priming of animals with DNA followed by protein booster generates heightened and mixed pro- and anti-inflammatory immune responses that are capable of

  13. The n-hexane and chloroform fractions of Piper betle L. trigger different arms of immune responses in BALB/c mice and exhibit antifilarial activity against human lymphatic filarid Brugia malayi.

    Science.gov (United States)

    Singh, Meghna; Shakya, Shilpy; Soni, Vishal Kumar; Dangi, Anil; Kumar, Nikhil; Bhattacharya, Shailja-Misra

    2009-06-01

    Modulation of immune functions by using herbal plants and their products has become fundamental regime of therapeutic approach. Piper betle Linn. (Piperaceae) is a widely distributed plant in the tropical and subtropical regions of the world and has been attributed as traditional herbal remedy for many diseases. We have recently reported the antifilarial and antileishmanial efficacy in the leaf extract of Bangla Mahoba landrace of P. betle which is a female plant. The present report describes the in vivo immunomodulatory efficacy of the crude methanolic extract and its n-hexane, chloroform, n-butanol fractions of the female plant at various dose levels ranging between 0.3 and 500 mg/kg in BALB/c. Attempts were also made to observe antifilarial activity of the active extracts and correlate it with the antigen specific immune responses in another rodent Mastomys coucha infected with human lymphatic filarial parasite Brugia malayi. The crude methanol extract and n-hexane fraction were found to potentiate significant (p<0.001) enhancement of both humoral (plaque forming cells, hemagglutination titre) as well as cell-mediated (lymphoproliferation, macrophage activation, delayed type hypersensitivity) immune responses in mice. The flow cytometric analysis of splenocytes of treated mice indicated enhanced population of T-cells (CD4(+), CD8(+)) and B-cells (CD19(+)). The n-hexane fraction (3 mg/kg) was found to induce biased type 2 cytokine response as revealed by increased IL-4(+) and decreased IFN-gamma(+) T-cell population while the chloroform fraction (10 mg/kg) produced a predominant type 1 cytokines. Crude methanolic extract (100 mg/kg) demonstrated a mixed type 1 and type 2 cytokine responses thus suggesting a remarkable immunomodulatory property in this plant. The induction of differential T-helper cell immune response appears ideal to overcome immunosuppression as observed in case of lymphatic, filarial Brugia malayi infection which may also be extended to other

  14. Physicochemical properties of the modeled structure of astacin metalloprotease moulting enzyme NAS-36 and mapping the druggable allosteric space of Heamonchus contortus, Brugia malayi and Ceanorhabditis elegans via molecular dynamics simulation.

    Science.gov (United States)

    Sharma, Om Prakash; Agrawal, Sonali; Kumar, M Suresh

    2013-12-01

    Nematodes represent the second largest phylum in the animal kingdom. It is the most abundant species (500,000) in the planet. It causes chronic, debilitating infections worldwide such as ascariasis, trichuriasis, hookworm, enterobiasis, strongyloidiasis, filariasis and trichinosis, among others. Molecular modeling tools can play an important role in the identification and structural investigation of molecular targets that can act as a vital candidate against filariasis. In this study, sequence analysis of NAS-36 from H. contortus (Heamonchus contortus), B. malayi (Brugia malayi) and C. elegans (Ceanorhabditis elegans) has been performed, in order to identify the conserved residues. Tertiary structure was developed for an insight into the molecular structure of the enzyme. Molecular Dynamics Simulation (MDS) studies have been carried out to analyze the stability and the physical properties of the proposed enzyme models in the H. contortus, B. malayi and C. elegans. Moreover, the drug binding sites have been mapped for inhibiting the function of NAS-36 enzyme. The molecular identity of this protease could eventually demonstrate how ex-sheathment is regulated, as well as provide a potential target of anthelmintics for the prevention of nematode infections.

  15. Genomes of parasitic nematodes (Meloidogyne hapla, Meloidogyne incognita, Ascaris suum and Brugia malayi) have a reduced complement of small RNA interference pathway genes: knockdown can reduce host infectivity of M. incognita.

    Science.gov (United States)

    Iqbal, Sadia; Fosu-Nyarko, John; Jones, Michael G K

    2016-07-01

    The discovery of RNA interference (RNAi) as an endogenous mechanism of gene regulation in a range of eukaryotes has resulted in its extensive use as a tool for functional genomic studies. It is important to study the mechanisms which underlie this phenomenon in different organisms, and in particular to understand details of the effectors that modulate its effectiveness. The aim of this study was to identify and compare genomic sequences encoding genes involved in the RNAi pathway of four parasitic nematodes: the plant parasites Meloidogyne hapla and Meloidogyne incognita and the animal parasites Ascaris suum and Brugia malayi because full genomic sequences were available-in relation to those of the model nematode Caenorhabditis elegans. The data generated was then used to identify some potential targets for control of the root knot nematode, M. incognita. Of the 84 RNAi pathway genes of C. elegans used as model in this study, there was a 42-53 % reduction in the number of effectors in the parasitic nematodes indicating substantial differences in the pathway between species. A gene each from six functional groups of the RNAi pathway of M. incognita was downregulated using in vitro RNAi, and depending on the gene (drh-3, tsn-1, rrf-1, xrn-2, mut-2 and alg-1), subsequent plant infection was reduced by up to 44 % and knockdown of some genes (i.e. drh-3, mut-2) also resulted in abnormal nematode development. The information generated here will contribute to defining targets for more robust nematode control using the RNAi technology.

  16. Brugia malayi Antigen (BmA Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells.

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    Emily E I M Mouser

    Full Text Available One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA and excretory-secretory product 62 (ES-62 from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs.

  17. AMPLIFICATION, CLONING AND SEQUENCE ANALYSIS OF CYSTEINE PROTEASE GENE FROM PERIODIC Brugia Malayi%周期型马来丝虫半胱氨酸蛋白酶基因的扩增、克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    黄为群; 方政; 陈阳; 姜声扬; 吴建军; 谢东方

    2007-01-01

    [目的]克隆周期型马来丝虫半胱氨酸蛋白酶(cysteine protease of periodic Brugia malayi,BmCP)基因到原核载体中,测定其序列,为进一步的研究奠定基础.[方法]从周期型马来丝虫虫体中抽提总RNA,以mRNA为模板,采用RT-PCR法体外扩增出BmCP基因序列,扩增产物经初步鉴定后将其克隆入pMD18-T载体,转化大肠杆菌(E. coli)DH5α,筛选阳性克隆,进行双酶切及PCR扩增签定,获得阳性重组质粒pMD18-T-BmCP,经测序验证,并进行同源性比较.[结果]RT-PCR扩增出一条约1 201 bp大小的特异性条带,重组质粒双酶切和以质粒为模板的PCR结果与预期相符,DNA序列分析与GeneBank已知的基因序列同源性为99%.[结论]成功构建了周期型马来丝虫半胱氨酸蛋白酶重组质粒pMD18-T克隆载体,为进一步研究该基因的功能提供了条件.

  18. 周期型马来丝虫复合基因重组质粒和相应表达蛋白的免疫学研究%Immunology comparision of composite gene recombinant plasmid and expressed protein of periodical Brugia malayi

    Institute of Scientific and Technical Information of China (English)

    王慧; 方政; 徐倩; 陆施娟; 钱一言; 徐怿琳; 方浩; 徐邦生

    2014-01-01

    粒/复合重组蛋白/CpG组小鼠淋巴细胞刺激增殖指数(1.629±0.235)高于复合重组蛋白组(1.248±0.110,P<0.05);免疫4、6周后,复合重组质粒/复合重组蛋白/CpG组和复合重组质粒/CpG组小鼠血清IFN-γ水平[(101.660±5.101)、(178.265±7.139)mg/L,(102.067±3.722)、(115.148±6.031)mg/L]均高于复合重组蛋白组[(75.438±2.102)、(82.004±3.777) mg/L,P均<0.05];免疫后6周,复合重组质粒/复合重组蛋白/CpG组和复合重组蛋白/CpG组的小鼠血清IL-4水平[(75.385±3.318)、(46.363±3.672)mg/L]均明显高于复合重组质粒/CpG组[(36.691±3.443)mg/L,P均<0.05).结论 pcDNA3.1-BmCPI/BmGAPDH核酸疫苗和相应蛋白疫苗均可诱导BALB/c小鼠产生特异性体液和细胞免疫应答反应.核酸疫苗-蛋白疫苗联合免疫效果有明显的优势.%Objective To construct a plasmid DNA vector expressing cysteine protease inhibitor and glyceraldehydes-3-phosphate dehydrogenase of periodic Brugia malayi(BmCPI/BmGAPDH),and purify the recombinant protein after transfecting the vector into human cervical carcinoma cells(Hela) for expression.To make a comparison of immunity efficacy between the recombinant plasmid and the homologous protein and to a lay theoretic and experimental basis for developing novel anti-filarial genetic engineering vaccines.Methods The amplified genes BmCPI and BmGAPDH and a plasmid vector were double enzymes digested and ligated to construct a recombinant plasmid pcDNA3.1 (+)-BmCPI/BmGAPDH,and this plasmid was transfected to Hela cells after being identified.G418 was used for screening transfectants,and the monoclonal resistant cell strain was determined by RT-PCR and SDS-PAGE.The recombinant protein was purified by affnity chromatography and identified by Western blotting.Sixty BALB/c mice were divided into 5 groups,12 per group,and they were immunized at 2,4,and 6 weeks.Mice in control groups were injected with PBS 100 μ1 or pcDNA3.1 100 μg/CpG 30 μg,and mice in

  19. Novel drug designing rationale againstBrugia malayi microfilariae using herbal extracts

    Institute of Scientific and Technical Information of China (English)

    SharmaRD; PetareS; ShindeGB; KalyanGoswami; ReddyMVR

    2010-01-01

    Objective:To explore the effect of herbal polyphenolics on filariasisin vitro.Methods: Two herbal extracts, methanolic extracts of roots ofVitex negundo Linn. (Nirgundi) and leaves ofAegle marmelos Juss. (Beal) in different concentrations ranging from40-80ng/mL were tested for their antifilarial activity either alone or in combination with diethyl carbonate (DEC)(300μg/mL) and/orH2O2 (0.5 mM).Results:Combination of DEC and each extract had significant anti-filarial effect. And fractions of both extracts were not effective as crude herbal extract.Conclusions:Such unique pharmacodynamics reported in this study might provide new drug development stratagem against filariasis.

  20. Detection of enzymes dehydrogenases and proteases inBrugia malayi filarial parasites.

    Science.gov (United States)

    Bhandary, Y P; Krithika, K N; Kulkarni, Sandeep; Reddy, M V R; Harinath, B C

    2006-03-01

    Lymphatic filariasis caused mainly by infection fromW. bancrofti andB. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis ofB. malayi mf, infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key enzymes of carbohydrate metabolism viz., Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose-6-phosphate dehydrogenase (G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high in mf lysate compared to their activity in lysates of the other two stages (PFlouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and the L(3) larval lysate had 6 protease molecules of 18, 25, 37, 49, 70 and 200 kDa size.

  1. Immune responses in human infections with Brugia malayi: specific cellular unresponsiveness to filarial antigens.

    Science.gov (United States)

    Piessens, W F; McGreevy, P B; Piessens, P W; McGreevy, M; Koiman, I; Saroso, J S; Dennis, D T

    1980-01-01

    We evaluated the cellular immune competence of 101 subjects living in an area of South Kalimantan (Borneo) where Malayan filariasis is endemic. All patients with elephantiasis but none with other clinical stages of filariasis reacted with adult worm antigens. The majority of subjects without clinical or parasitological evidence of filariasis and approximately one-half of those with amicrofilaremic filariasis reacted with microfilarial antigens. In contrast, most patients with patent microfilaremia did not respond to microfilarial antigens. The in vitro reactivity of all patient categories to nonparasite antigens was similar to that of the distant control group. These results indicate that patent microfilaremia is associated with a state of specific cellular immune unresponsiveness and are consistent with the current hypothesis that the various clinical manifestations of filariasis result from different types of immune responses to distinct antigens associated with different developmental stages of filarial worms. PMID:7350196

  2. Brugia malayi infective larvae fail to activate Langerhans cells and dermal dendritic cells in human skin.

    Science.gov (United States)

    Cotton, R N; McDonald-Fleming, R; Boyd, A; Spates, K; Nutman, T B; Tolouei Semnani, R

    2015-02-01

    Filarial infection in humans is initiated when a mosquito deposits third-stage parasite larvae (L3) in the skin. Langerhans cells (LCs) and dermal dendritic cells (DDCs) are the first cells that the parasite encounters, and L3s must evade these highly effective antigen-presenting cells to establish infection. To assess LC and DDC responses to L3 in human skin, we employed three models of increasing physiologic relevance: in vitro-generated LCs, epidermal blister explants and full-thickness human skin sections. In vitro-generated LCs expressed TLR1-10 and robustly produced IL-6 and TNF-α in response to PolyI:C, but pre-exposure to L3s did not alter inflammatory cytokine production or TLR expression. L3s did not modulate expression of LC markers CDH1, CD207, or CD1a, or the regulatory products TSLP or IDO in epidermal explants or in vitro-generated LC. LC, CD14+ DDC, CD1c+ DC and CD141+ DC from human skin sections were analysed by flow cytometry. While PolyI:C potently induced CCL22 production in LC, CD1c+ DC, and CD141+ DC, and IL-10 production in LC, L3s did not modulate the numbers of or cytokine production by any skin DC subset. L3s broadly failed to activate or modulate LCs or DDCs, suggesting filarial larvae expertly evade APC detection in human skin.

  3. Proinflammatory Cytokine Gene Expression by Murine Macrophages in Response to Brugia malayi Wolbachia Surface Protein

    Directory of Open Access Journals (Sweden)

    Chantima Porksakorn

    2007-01-01

    Full Text Available Wolbachia, an endosymbiotic bacterium found in most species of filarial parasites, is thought to play a significant role in inducing innate inflammatory responses in lymphatic filariasis patients. However, the Wolbachia-derived molecules that are recognized by the innate immune system have not yet been identified. In this study, we exposed the murine macrophage cell line RAW 264.7 to a recombinant form of the major Wolbachia surface protein (rWSP to determine if WSP is capable of innately inducing cytokine transcription. Interleukin (IL-1β, IL-6, and tumor necrosis factor (TNF mRNAs were all upregulated by the rWSP stimulation in a dose-dependant manner. TNF transcription peaked at 3 hours, whereas IL-1β and IL-6 transcription peaked at 6 hours post-rWSP exposure. The levels of innate cytokine expression induced by a high-dose (9.0 μg/mL rWSP in the RAW 264.7 cells were comparable to the levels induced by 0.1 μg/mL E. coli-derived lipopolysaccharides. Pretreatment of the rWSP with proteinase-K drastically reduced IL-1β, IL-6, and TNF transcription. However, the proinflammatory response was not inhibited by polymyxin B treatment. These results strongly suggest that the major Wolbachia surface protein molecule WSP is an important inducer of innate immune responses during filarial infections.

  4. Tissue and stage-specific distribution of Wolbachia in Brugia malayi.

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    Kerstin Fischer

    2011-05-01

    Full Text Available BACKGROUND: Most filarial parasite species contain Wolbachia, obligatory bacterial endosymbionts that are crucial for filarial development and reproduction. They are targets for alternative chemotherapy, but their role in the biology of filarial nematodes is not well understood. Light microscopy provides important information on morphology, localization and potential function of these bacteria. Surprisingly, immunohistology and in situ hybridization techniques have not been widely used to monitor Wolbachia distribution during the filarial life cycle. METHODS/PRINCIPAL FINDINGS: A monoclonal antibody directed against Wolbachia surface protein and in situ hybridization targeting Wolbachia 16S rRNA were used to monitor Wolbachia during the life cycle of B. malayi. In microfilariae and vector stage larvae only a few cells contain Wolbachia. In contrast, large numbers of Wolbachia were detected in the lateral chords of L4 larvae, but no endobacteria were detected in the genital primordium. In young adult worms (5 weeks p.i., a massive expansion of Wolbachia was observed in the lateral chords adjacent to ovaries or testis, but no endobacteria were detected in the growth zone of the ovaries, uterus, the growth zone of the testis or the vas deferens. Confocal laser scanning and transmission electron microscopy showed that numerous Wolbachia are aligned towards the developing ovaries and single endobacteria were detected in the germline. In inseminated females (8 weeks p.i. Wolbachia were observed in the ovaries, embryos and in decreasing numbers in the lateral chords. In young males Wolbachia were found in distinct zones of the testis and in large numbers in the lateral chords in the vicinity of testicular tissue but never in mature spermatids or spermatozoa. CONCLUSIONS: Immunohistology and in situ hybridization show distinct tissue and stage specific distribution patterns for Wolbachia in B. malayi. Extensive multiplication of Wolbachia occurs in the lateral chords of L4 and young adults adjacent to germline cells.

  5. Rapid Diagnosis of Brugia malayi and Wuchereria bancrofti Filariasis by an Acridine Orange/Microhematocrit Tube Technique

    Science.gov (United States)

    1990-01-01

    cyte monophenol oxida uviN in mos- thme laboratory biology and mai nance of-ti-des quitoescexposed to microfil ac ofiof’ aria mm. fri vilfofus. Mosquito...h aaie r detected in samples diluted to alevel ofappr-dmately 18;Rcmne l,18) h aaie r 50/mI. K \\/i )’c cl stained by the acridine orange dye and can...Ridley, Department of ratory Medicin, College of teninary Medicine; *Division of Biology and ji~epartment of Anatomy anid Phys , College of Veterinary

  6. DETECTION OF BRUGIA MALAYI INFECTED MOSQUITOES WITH SPECIES SPECIFIC DNA PROBE pBm 15, IN RIAU, INDONESIA

    Directory of Open Access Journals (Sweden)

    L. Kurniawan

    2012-09-01

    Full Text Available A species specific DNA probe (pBm15 was used in a field area where 2 filarial infections coexist: B.malayi in man and B.pahangi in cats. In our laboratory in Jakarta, this DNA probe proved to be sensitive enough to detect 500 ng DNA. One to two infective larvae of B.malayi could be detected with ease. This DNA probe did not react with infective larvae of wuchereria bancrofti, B.pahangi, and Dirofilaria spp. Non specific binding caused by undefined mosquito components was overcome with proteinase K and chitinase treatment. This additional step, made it possible for whole body mosquitoes to be squashed directly onto nitrocellulose paper. A comparative study of experimental infections of laboratory bred mosquitoes infected with B.malayi, showed no difference in infection rate between the group examined by dissection or by DNA probing. Mosquitoes which are vectors in Riau were collected and fed on microfilaremic patients of Riau. The set of mosquitoes were tested in parallel with mosquitoes infected with B.pahangi from cats. All fed mosquitoes were tested after 10-12 days. Only mosquitoes infected with B.malayi reacted in the assay. This study shows a success in applying the DNA probe technique in Jakarta. Further application in the field should be encouraged, with some modification of the DNA probing technique, for cheaper and easier implementation.

  7. Attempts to Image the Early Inflammatory Response during Infection with the Lymphatic Filarial Nematode Brugia pahangi in a Mouse Model

    Science.gov (United States)

    Ritchie, Ryan; Goundry, Amy; O’Neill, Kerry; Marchesi, Francesco; Devaney, Eileen

    2016-01-01

    Helminth parasites remain a major constraint upon human health and well-being in many parts of the world. Treatment of these infections relies upon a very small number of therapeutics, most of which were originally developed for use in animal health. A lack of high throughput screening systems, together with limitations of available animal models, has restricted the development of novel chemotherapeutics. This is particularly so for filarial nematodes, which are long-lived parasites with a complex cycle of development. In this paper, we describe attempts to visualise the immune response elicited by filarial parasites in infected mice using a non-invasive bioluminescence imaging reagent, luminol, our aim being to determine whether such a model could be developed to discriminate between live and dead worms for in vivo compound screening. We show that while imaging can detect the immune response elicited by early stages of infection with L3, it was unable to detect the presence of adult worms or, indeed, later stages of infection with L3, despite the presence of worms within the lymphatic system of infected animals. In the future, more specific reagents that detect secreted products of adult worms may be required for developing screens based upon live imaging of infected animals. PMID:27992545

  8. Gene : CBRC-TTRU-01-0063 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available AMILY PROTEIN [Brugia malayi] gb|EDP39170.1| PPE FAMILY PROTEIN, putative [Brugia malayi] 6e-73 33% METVHMVKVPRVTLPMVTVLMVT...MPMVMVLMVKLPIVTVPMVIVPVVTVLTVKVPMVTLPTVITLKVTELTVIVAMLTVPLLRVLMVILLTVTVPMVRVLMLRLLMMTTLMVAVPMVT...MLIVAVLMVIVLMLTVLPVTLSMVSVSMVTVLTMILLTVAVLLVTLLRVTVCTVPMVSVPTVTFPMVTVPTVTVSMVRVCMETVHIVTFPTVKMPMVT...VLTMTVPMVTVPMVTVPMVIVPMVTVLMVTVPMVRVLMVTVPMVTVTTVTMPMLTVLTIKMLIVTVPMVILPMGSVLTVKVPMVTLPTVIVHKVRVLTVLVPMVT...VPMVAVPMVTVPMVIVLMVIEPMVTVPMVAVPMVTMFTVSMPMVTLLMVTVLTVKVLTVLVLTVIVLMVIAPW ...

  9. Immunization with Brugia malayi Myosin as Heterologous DNA Prime Protein Boost Induces Protective Immunity against B. malayi Infection in Mastomys coucha

    Science.gov (United States)

    Gupta, Jyoti; Misra, Sweta; Misra-Bhattacharya, Shailja

    2016-01-01

    The current control strategies employing chemotherapy with diethylcarbamazine, ivermectin and albendazole have reduced transmission in some filaria-endemic areas, there is growing interest for complementary approaches, such as vaccines especially in light of threat of parasite developing resistance to mainstay drugs. We earlier demonstrated recombinant heavy chain myosin of B. malayi (Bm-Myo) as a potent vaccine candidate whose efficacy was enhanced by heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) vaccination in BALB/c mice. BALB/c mouse though does not support the full developmental cycle of B. malayi, however, the degree of protection may be studied in terms of transformation of challenged infective larvae (L3) to next stage (L4) with an ease of delineating the generated immunological response of host. In the current investigation, DNA vaccination with Bm-Myo was therefore undertaken in susceptible rodent host, Mastomys coucha (M. coucha) which sustains the challenged L3 and facilitates their further development to sexually mature adult parasites with patent microfilaraemia. Immunization schedule consisted of Myo-pcD and Myo-pcD+Bm-Myo followed by B. malayi L3 challenge and the degree of protection was evaluated by observing microfilaraemia as well as adult worm establishment. Myo-pcD+Bm-Myo immunized animals not only developed 78.5% reduced blood microfilarial density but also decreased adult worm establishment by 75.3%. In addition, 75.4% of the recovered live females revealed sterilization over those of respective control animals. Myo-pcD+Bm-Myo triggered higher production of specific IgG and its isotypes which induced marked cellular adhesion and cytotoxicity (ADCC) to microfilariae (mf) and L3 in vitro. Both Th1 and Th2 cytokines were significantly up-regulated displaying a mixed immune response conferring considerable protection against B. malayi establishment by engendering a long-lasting effective immune response and therefore emerges as a potential vaccination method against LF. PMID:27828973

  10. Possible implication of oxidative stress in anti filarial effect of certain traditionally used medicinal plants in vitro against Brugia malayi microfilariae

    Directory of Open Access Journals (Sweden)

    R D Sharma

    2010-01-01

    Full Text Available Introduction: Tropical disease research scheme of World Health Organization has duly recognized traditional medicine as alternative for antifilarial drug development. Polyphenolic compounds present in traditionally used herbal medicines are natural antioxidants; however, paradoxically they may exert pro-oxidant effect. Popular drug diethyl carbamazine citrate harnesses the innate inflammatory response and the consequent oxidative assault to combat invading microbes. Methods: With this perspective, extracts of Vitex negundo L. (roots, Butea monosperma L. (leaves, Aegle marmelos Corr. (leaves, and Ricinus communis L. (leaves were selected to explore the possible role of oxidative rationale in the antifilarial effect in vitro. Results: Apart from the last, other three plant extracts were reported to have polyphenolic compounds. Dose-dependent increase was found in the levels of lipid peroxidation and protein carbonylation for all the three plant extracts except Ricinus communis L. (leaves. Such increase in oxidative parameters also showed some degree of plant-specific predilection in terms of relatively higher level of particular oxidative parameter. High degree of correlation was observed between the antifilarial effect and the levels of corresponding oxidative stress parameters for these three plants. However, extracts of Ricinus communis L. (leaves which is relatively deficient in polyphenolic ingredients recorded maximum 30% loss of motility and also did not show any significant difference in various stress parameters from corresponding control levels. Conclusion: These results reveal that targeted oxidative stress might be crucial in the pharmacodynamics.

  11. NCBI nr-aa BLAST: CBRC-TTRU-01-1053 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1053 ref|XP_001901891.1| endoplasmic reticulum multispan transmembrane... protein [Brugia malayi] gb|EDP29450.1| endoplasmic reticulum multispan transmembrane protein, putative [Brugia malayi] XP_001901891.1 0.15 23% ...

  12. NCBI nr-aa BLAST: CBRC-TTRU-01-0887 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0887 ref|XP_001902801.1| Transmembrane amino acid transporter protein ...[Brugia malayi] gb|EDP28347.1| Transmembrane amino acid transporter protein [Brugia malayi] XP_001902801.1 0.065 28% ...

  13. NCBI nr-aa BLAST: CBRC-CREM-01-1342 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1342 ref|YP_198592.1| Predicted permease [Wolbachia endosymbiont strai...n TRS of Brugia malayi] gb|AAW71350.1| Predicted permease [Wolbachia endosymbiont strain TRS of Brugia malayi] YP_198592.1 2e-05 25% ...

  14. NCBI nr-aa BLAST: CBRC-FCAT-01-1234 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FCAT-01-1234 ref|YP_198404.1| 50S ribosomal protein L13 [Wolbachia endosymbion...t strain TRS of Brugia malayi] gb|AAW71162.1| Ribosomal protein L13 [Wolbachia endosymbiont strain TRS of Brugia malayi] YP_198404.1 3.4 26% ...

  15. Og4C3 circulating antigen, anti-Brugia malayi IgG and IgG4 titers in Wuchereria bancrofti infected patients, according to their parasitological status.

    Science.gov (United States)

    Chanteau, S; Glaziou, P; Luquiaud, P; Plichart, C; Moulia-Pelat, J P; Cartel, J L

    1994-09-01

    This study involved 221 microfilaremic (Mf+), 302 amicrofilaremic (Mf-) antigen positive (AG+) and 1454 Mf-antigen negative (AG-) individuals living in endemic villages. Whatever the group considered, antigen and antibody titers were widely distributed. Og4C3 antigen, detected both in Mf- and Mf+ patients, was significantly higher in Mf+ patients. The Mf parasitological status did not significantly influence the antifilarial antibodies levels in the infected AG+ individuals, although IgG4 was more discriminant. In the supposedly uninfected individuals (Mf-AG-), anti-filarial IgG and IgG4 could be detected in a large proportion of the group. Og4C3 circulating antigen test was confirmed to be a good marker of active Wuchereria bancrofti infection.

  16. Lymphatic Filariasis

    Science.gov (United States)

    ... of obtaining a "blood meal." Laboratory strains of Aedes aegypti can be infected with Brugia. Credit: DPDx , PHIL General Information Most common ... & Control How to stay healthy or get better... ...

  17. Dicty_cDB: Contig-U15773-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ypti infected with Brugia Mala... 48 2.0 1 ( DV349458 ) NABXO63TO Aedes aegypti infected with Plasmodium ...... 48 2.0 1 ( DV349457 ) NABXO63TF Aedes aegypti infected with Plasmodium ... 48 2.

  18. Midgut barrier imparts selective resistance to filarial worm infection in Culex pipiens pipiens.

    Directory of Open Access Journals (Sweden)

    Michelle L Michalski

    Full Text Available Mosquitoes in the Culex pipiens complex thrive in temperate and tropical regions worldwide, and serve as efficient vectors of Bancroftian lymphatic filariasis (LF caused by Wuchereria bancrofti in Asia, Africa, the West Indies, South America, and Micronesia. However, members of this mosquito complex do not act as natural vectors for Brugian LF caused by Brugia malayi, or for the cat parasite B. pahangi, despite their presence in South Asia where these parasites are endemic. Previous work with the Iowa strain of Culex pipiens pipiens demonstrates that it is equally susceptible to W. bancrofti as is the natural Cx. p. pipiens vector in the Nile Delta, however it is refractory to infection with Brugia spp. Here we report that the infectivity barrier for Brugia spp. in Cx. p. pipiens is the mosquito midgut, which inflicts internal and lethal damage to ingested microfilariae. Following per os Brugia exposures, the prevalence of infection is significantly lower in Cx. p. pipiens compared to susceptible mosquito controls, and differs between parasite species with <50% and <5% of Cx. p. pipiens becoming infected with B. pahangi and B. malayi, respectively. When Brugia spp. mf were inoculated intrathoracically to bypass the midgut, larvae developed equally well as in controls, indicating that, beyond the midgut, Cx. p. pipiens is physiologically compatible with Brugia spp. Mf isolated from Cx. p. pipiens midguts exhibited compromised motility, and unlike mf derived from blood or isolated from the midguts of Ae. aegypti, failed to develop when inoculated intrathoracically into susceptible mosquitoes. Together these data strongly support the role of the midgut as the primary infection barrier for Brugia spp. in Cx. p. pipiens. Examination of parasites recovered from the Cx. p. pipiens midgut by vital staining, and those exsheathed with papain, suggest that the damage inflicted by the midgut is subcuticular and disrupts internal tissues. Microscopic studies

  19. GenBank blastx search result: AK289222 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK289222 J100067E05 AF030944.1 AF030944 Brugia malayi microfilarial sheath protein ...SHP3a (Bmshp3a) and microfilarial sheath protein SHP3 precursor (Bmshp3) genes, complete cds. INV 0.0 0 ...

  20. AcEST: DK953053 [AcEST

    Lifescience Database Archive (English)

    Full Text Available .18 sp|Q01202|MYSP_BRUMA Paramyosin OS=Brugia malayi PE=2 SV=2 33 1.6 sp|Q11IG3|Y1416_MESSB UPF0283 membrane...ct: 168 DQLKKDKHLAEKAAERFEAQTVELSNKVEDLNRHVNDLAQQRQRLQAENNDL 219 >sp|Q11IG3|Y1416_MESSB UPF0283 membrane pro

  1. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    Directory of Open Access Journals (Sweden)

    Catherine B Poole

    Full Text Available In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

  2. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    Science.gov (United States)

    Poole, Catherine B; Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C; Carlow, Clotilde K S

    2012-01-01

    In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

  3. AcEST: DK949163 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 36 2.3 tr|A8P3K7|A8P3K7_BRUMA Retinitis pigmentosa GTPase regulator-lik... 35 3.... 92 FGGGGN 97 >tr|A8P3K7|A8P3K7_BRUMA Retinitis pigmentosa GTPase regulator-like protein OS=Brugia malayi GN

  4. Repurposing auranofin as a lead candidate for treatment of lymphatic filariasis and onchocerciasis.

    Directory of Open Access Journals (Sweden)

    Christina A Bulman

    2015-02-01

    Full Text Available Two major human diseases caused by filariid nematodes are onchocerciasis, or river blindness, and lymphatic filariasis, which can lead to elephantiasis. The drugs ivermectin, diethylcarbamazine (DEC, and albendazole are used in control programs for these diseases, but are mainly effective against the microfilarial stage and have minimal or no effect on adult worms. Adult Onchocerca volvulus and Brugia malayi worms (macrofilariae can live for up to 15 years, reproducing and allowing the infection to persist in a population. Therefore, to support control or elimination of these two diseases, effective macrofilaricidal drugs are necessary, in addition to current drugs. In an effort to identify macrofilaricidal drugs, we screened an FDA-approved library with adult worms of Brugia spp. and Onchocerca ochengi, third-stage larvae (L3s of Onchocerca volvulus, and the microfilariae of both O. ochengi and Loa loa. We found that auranofin, a gold-containing drug used for rheumatoid arthritis, was effective in vitro in killing both Brugia spp. and O. ochengi adult worms and in inhibiting the molting of L3s of O. volvulus with IC50 values in the low micromolar to nanomolar range. Auranofin had an approximately 43-fold higher IC50 against the microfilariae of L. loa compared with the IC50 for adult female O. ochengi, which may be beneficial if used in areas where Onchocerca and Brugia are co-endemic with L. loa, to prevent severe adverse reactions to the drug-induced death of L. loa microfilariae. Further testing indicated that auranofin is also effective in reducing Brugia adult worm burden in infected gerbils and that auranofin may be targeting the thioredoxin reductase in this nematode.

  5. ASPEK ZOONOTIK PARASIT NEMATODA PADA KERA DAN BINATANG MENGERAT DI BENGKULU, SUMATERA. INDONESIA

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    Untung S.

    2012-09-01

    Full Text Available Twentyfive monkeys and 481 rats were examined for parasitic nematodes in Bengkulu, nine species of nematode were found infecting these animals. Five of filarían nematodes, i.e. Brugia malayi, Brugia pahangi, Dirofilaria magnilarvatum and Edesonfilaria malayensis were infecting monkeys and one speciesTBreinlia booliati, was found infecting rats. Three species of gastrointestinal helminths, i.e. Trichuris trichiura, Enterobius vermicularis and Oestophagomomum spp were found in monkeys; a lung worm, Angiostrongylus cantonensis, was found in rats. The most important nematode species is B. malayi, which was found in Presbytis cristatus (36.8 % and in Macaca fascicularis (20.0 %. T. trichiura was found in R. cristatus (47.9 % and A. cantonensis in Rattus argentiventer (4.0 % and Rattus tiomanicus (2.9%.

  6. Evaluating Vaccine Candidates for Filariasis

    Science.gov (United States)

    2013-04-16

    of IgE antibodies can then induce type 1 hypersensitivities upon exposure to worm antigens. This is not just a theoretical problem; this specific...Cat 15-040-CV), 1% glucose (Sigma- Aldrich Co. St. Louis, MO), 100 I.U/ml Penicillin (Mediatech inc.), 100 μg/ml streptomycin (Mediatech inc), and...Casey HW. 1990. Brugia pahangi: effects of duration of infection and parasite burden on lymphatic lesion severity, granulomatous hypersensitivity , and

  7. Excretory-secretory and somatic antigens in the diagnosis of human filariasis.

    Science.gov (United States)

    Kaushal, N A; Hussain, R; Ottesen, E A

    1984-06-01

    In order to compare the immunodiagnostic value of excretory-secretory (E-S) antigens derived from adult Brugia malayi worms with somatic antigens derived from adults, microfilariae (Mf) and infective larvae (L3) of these parasites, well defined serum pools from patients with filarial (brugia, bancrofti, loa and perstans) and non-filarial (ascaris, stronglyoides, toxocara, echinococcus, cysticercus and schistosoma) helminth infections were tested against antigens derived from these different life cycle stages of B. malayi in a Staphylococcus aureus radioimmunoprecipitation assay (S. aureus RIA). The adult brugia antigens proved significantly more discriminatory than those of the other parasite stages, with the homologous brugia serum pool also showing greater reactivity to adult than to L3 and Mf antigens. Similar results were obtained when individual sera from patients (rather than serum pools) were tested in the same assay. The most surprising finding was the minimal reactivity seen between the adult filarial antigens and the non-filarial serum pools despite the presence in these pools of strong antibody reactivity with their homologous antigens. The reasons underlying the unexpected specificity of this S. aureus RIA for discriminating among sera from filarial and non-filarial infections were analysed qualitatively by immunoprecipitation techniques. It was found that use of the chloramine-T method for radioiodination resulted in preferential labelling of the low molecular weight (mol. wt) proteins (10-70,000 daltons) in the B. malayi adult somatic antigen and that these antigens were bound primarily by the filarial and not the non-filarial serum pools. These findings suggest that lower mol. wt helminth antigens may show greater species specificity than those with higher mol. wt, and those with higher mol. wt, greater cross-reactivity. If substantiated by further analysis, such results would have important implications for the subsequent isolation of diagnostically

  8. ECOLOGY AND INFECTION RATES OF NATURAL VECTORS OF FILARIASIS IN TANAH INTAN, SOUTH KALIMANTAN (BORNEO), INDONESIA

    OpenAIRE

    Soeroto Atmosoedjono; Purnomo Purnomo; Sutanti Ratiwayanto; Harijani A. Marwoto; Bangs, Michael J.

    2012-01-01

    Data ekologi nyamuk vektor dan tingkat infeksi filaría secara alami dan secara buatan telah diperoleh dari perkebunan karet di Kalimantan Selatan, Indonesia. Berbagai macam cara penangkapan dalam kondisi ekologi yang berbeda telah dipakai dalam pengumpulan 51 jenis nyamuk (N = 95.735). Pembedahan nyamuk, infeksi buatan dan identifikasi larva filaría mengikuti prosedur dan kunci yang sudah baku. Infeksi filaría Brugia, Breinlia dan Cardiofílaria secara alami ditemukan pada nyamuk Coquillettidi...

  9. Lymphedema secondary to filariasis.

    Science.gov (United States)

    Leonard, J C; Humphrey, G B; Basmadjian, G

    1985-03-01

    A 1-year-old immunodeficient boy developed brawny edema of the left foot. Lymphoscintigraphy revealed no evidence of left inguinal activity following pedal injection of Tc-99m-Sn phosphate. Over the next two months, the patient developed lymphedema on the right and repeat scintigraphy demonstrated no movement of isotope from the dorsum of either foot. Subsequent studies identified microfilaria in a nocturnal blood smear, which were thought to represent Brugia beaveri acquired by mosquito transmission in Oklahoma.

  10. Lymphedema secondary to filariasis

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.C.; Humphrey, G.B.; Basmadjian, G.

    1985-03-01

    A 1-year-old immunodeficient boy developed brawny edema of the left foot. Lymphoscintigraphy revealed no evidence of left inguinal activity following pedal injection of Tc-99m-Sn phosphate. Over the next two months, the patient developed lymphedema on the right and repeat scintigraphy demonstrated no movement of isotope from the dorsum of either foot. Subsequent studies identified microfilaria in a nocturnal blood smear, which were thought to represent Brugia beaveri acquired by mosquito transmission in Oklahoma.

  11. ROLE OF FINE NEEDLE ASPIRATION CYTOLOGY (FNAC IN DIAGNOSIS OF ASYMPTOMATIC MICROFILARIASIS

    Directory of Open Access Journals (Sweden)

    Reena

    2015-05-01

    Full Text Available Filariasis is a tropical and subtropical disease caused by Wuchereria Bancrofti and Brugia Malayi and transmitted by Culex mosquito. Lymphatic Filariasis is a major health problem in countries like India, China, Indonesia, and Africa. Diagnosis of Filari a is done by conventional methods like peripheral blood smear examination, Fluorescent capillary method and filarial antigen detection by Rapid card method. Here we present four unusual cases with swellings presented in surgical outdoor and referred for FN AC. Our aim is to evaluate and emphasize the utility and importance of Fine Needle Aspiration in diagnosing Microfilarasis in clinically unsuspected cases.

  12. SURVEI DARAH JARI FILARIASIS DI DESA BATUMARTA X KEC. MADANG SUKU III KABUPATEN OGAN KOMERING ULU (OKU TIMUR, SUMATERA SELATAN TAHUN 2012

    Directory of Open Access Journals (Sweden)

    R. Irpan Pahlepi

    2015-01-01

    Full Text Available AbstrakFilariasis atau penyakit kaki gajah adalah golongan penyakit menular yang disebabkan oleh cacing filaria  yang  ditularkan  melalui  berbagai  jenis  nyamuk.  Penyebaran  filariasis  hampir  meliputi  seluruh wilayah di Indonesia termasuk Kabupaten Ogan Komering Ulu (OKU Timur. Angka kesakitan filarisis di Kabupaten OKU Timur tahun 2007 sebesar 1,05%. Kegiatan pengobatan massal di Kabupaten OKU Timur belum pernah dilakukan sampai saat ini, sehingga perlu dilakukan penelitian yang bertujuan untuk mengetahui tingkat penyebaran penyakit filariasis. Penelitian ini merupakan penelitian survei dengan desain potong lintang. Pengambilan dan pemeriksaan sediaan darah jari dilakukan pada malam hari dimulai pukul 19.00 WIB. Jumlah penduduk yang diperiksa sebanyak 502. Hasil pemeriksaan diperoleh 4 orang positif mikrofilaria (Mf_ rate 0,8% dengan spesies Brugia  malayi  dan  kepadatan  rata-rata  200mf/ml.  Seluruh  kasus  yang  ditemukan  merupakan  kasus baru. Hasil penelitian ini menunjukkan bahwa penularan filariasis masih terjadi di Kabupaten OKU Timur sehingga perlu adanya pengobatan massal untuk mencegah penularan lebih lanjut.Kata kunci : Filariasis, Brugia malayi, Survei darah jari, OKU TimurAbstractFilariasis or elephantiasis is an infectious diseases caused by filarial worms that transmitted by various species of mosquitoes. Filariasis distributions almost covers all districts in Indonesia including East Ogan Komering Ulu (OKU. Filarisais morbidity in East OKU regency in 2007 was 1.05 %. Mass treatment in the district of East OKU have not been done yet, so it is necessary to do a research that aim to determine the prevalen of filariasis. This study is a cross-sectional survey design. Collection and examination of finger’s blood was done at night starting at 19:00. Number of people examined were 502. Examination results obtained 4 positive microfilariae (Mf_ rate 0.8 % of Brugia malayi and the average density of 200

  13. GAMBARAN PENULARAN FILARIASIS DI PROVINSI SULAWESI BARAT

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    Sitti Chadijah

    2014-10-01

    Full Text Available AbstractFilariasis is a zoonotic disease transmitted by mosquito that can cause disability. This study was aimed to identify filariasis transmission area in West Sulawesi. This cross sectional study was carried out from March to Novemer 2011. Sample was selected people in the endemic area (where chronic case was found in Mamuju, North Mamuju, and Mamasa District, West Sulawesi. Data were collected through night blood survey (started at 8 pm and entomology survey. Microfilariae was found in Polewali (microfilaria rate 2,81% and Wulai Village (microfilaria rate 2,06% in Bambalamotu sub-district (microfilaria rate 1,45%, North Mamuju District. Brugia malayi was found in the survey area and Anopheles barbirostrisas vector. North Mamuju is a filariasis endemic area.Keywords: filariasis, Brugia malayi, Anopheles barbirostris.AbstrakFilariasis adalah penyakit zoonosis yang ditularkan melalui gigitan nyamuk yang dapat menyebabkan  kecatatan.  Tujuaan  penelitian  ini  adalah  mengidentifikasi  daerah  penularan filariasis di provinsi Sulawesi Barat. Penelitian ini dilakukan dari bulan Maret sampai Nopember 2011 dengan disain penelitian observasional menggunakan rancangan potong lintang. Sampel adalah masyarakat di desa endemis filariasis (terdapat kasus kronis yang terpilih di Kabupaten Mamuju, Kabupaten Mamuju Utara, dan Kabupaten Mamasa, Sulawesi Barat. Kegiatan yang dilakukan yaitu Survei Darah Jari pada malam hari mulai pukul 20.00, dan survei entomologi filariasis. Hasil menunjukkan bahwa sediaan darah positif mengandung mikrofilaria ditemukan di  Desa  Polewali  (mikrofilaria  rate  2,81%  dan  Dusun  Wulai  (mikrofilaria  rate  2,06%  di Kecamatan Bambalamotu (mikrofilaria rate 1,45%, Kabupaten Mamuju Utara. Cacing filaria yang  ditemukan  adalah  Brugia  malayi  dengan  vektornya  adalah Anopheles  barbirostris. Disimpulkan bahwa Kabupaten Mamuju Utara adalah daerah endemis filariasis.Kata kunci: filariasis, Brugia

  14. Serum antibody responses to Wolbachia surface protein in patients with human lymphatic filariasis.

    Science.gov (United States)

    Shiny, Chandanapurath; Krushna, Nagampalli S A; Archana, Bairavasundaram; Farzana, Begum; Narayanan, Rangarajan B

    2009-12-01

    Wolbachia surface protein (WSP), which is the most abundantly expressed protein of Wolbachia from the human filarial parasite Brugia malayi, was chosen for the present study. B-cell epitope prediction of the WSP protein sequence indicates a high antigenicity, surface probability and hydrophilicity by DNA STAR software analysis. ProPred analysis suggests the presence of HLA class II binding regions in the WSP protein that contribute to T-cell responses and isotype reactivity. In order to validate these findings, the gene coding for endosymbiont WSP was PCR-amplified from the genomic DNA of the human filarial parasite Brugia malayi and cloned in T-7 expression vector pRSET-A. Western blot and ELISA at the total IgG level with recombiant WSP indicated a significantly elevated reactivity in CP compared to MF, EN and NEN individuals. Isotype ELISA also suggested an elevated reactivity in CP patients at the IgG1 level. In contrast, WSP-specific IgG4 levels were found to be elevated in MF patients compared to CP and EN. Besides this, WSP-specific IgE levels indicated an elevated reactivity in CP and MF patients compared to normals. Observations from ELISA supported the in silico predictions that indicate the presence of B- and T-cell epitopes. Hence, a combinatorial approach of in silico predictions and wet-lab studies provides interesting insights into the role of Wolbachia proteins in filarial pathogenesis.

  15. BIOTECHNOLOGY OF THE FILARIA OF INDONESIA

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    F. Partono

    2012-09-01

    Full Text Available More than 90 million people were currently infected with lymphatic filariasis and mo-third of them lived in China, India and Indonesia. Filariasis is endemic throughout the entire Indonesian Archipelago. More than 20 million people lived in endemic areas and 3-4 million people were estimated to have the infection. Control measures have reduced the prevalence of infections in some areas, but the disease remained a public health problem in many outer islands of Indonesia. Recent development in monoclonal antibodies and recombinant technology of DNA have made it possible to apply these new tools in the studies of filariasis, and three groups in Indonesia are currently using these new technology. The studies with the Imperial College of London will be presented by Dr. Rick Maizels. Collaboration with the New England Biolabs and Smith College involve the use of a stage and species-specific monoclonal antibody against the infective larvae of Brugia malayi, a double blind comparison of conventional methods and DNA probes for the diagnosis of brugian filariasis, and phylogentic studies of the brugian parasites. The ELISA using the monoclonal antibody has been adapted for field use in Jakarta. It is simple to use, does not cross-react with the infective stage of Brugia pahangi, but does so with the infective stage of the non-sympatric Brugia timori. The reagent is useful to acurately monitor the progress of control programs in endemic areas of brugian filariasis. The oligonucleotide DNA probes for B.malayi and B.pahangi were both qualitatively and quantitatively comparable to the conventional methods for the diagnosis of brugian parasites in cats and man. Sequencing data of the repeated DNA sequences of various brugian parasites indicated their homologies and divergences. The anthropophilic strain of B.malayi and B.timori showed similarity in their biological characteristics and repeated DNA sequences and they are phylogenically probably closely related

  16. The lethal effects of the cibarial and pharyngeal armatures of mosquitoes on microfilariae.

    Science.gov (United States)

    McGreevy, P B; Bryan, J H; Oothuman, P; Kolstrup, N

    1978-01-01

    Microfilariae of Wuchereria bancrofti and Brugia pahangi were killed by the chewing action of the cibarial and pharyngeal armatures and other papillae and spines in the fore-gut of mosquitoes. The proportion of ingested microfilariae that were killed was largely dependent on the presence and shape of the cibarial armature. Anopheles farauti No. 1 and Anopheles gambiae species A and B have well developed cibarial armatures and killed 36 to 96% of the ingested microfilariae. Culex pipiens fatigans has a poorly developed cibarial armature and killed only 6% of the microfilariae. Aedes aegypti and Aedes togoi lack cibarial armatures but have the remaining fore-gut structures. They killed only 2 to 22% of the microfilariae. The significance of these observations in relation to the control of filariasis with diethylcarbamazine is discussed.

  17. Wuchereria bancrofti filariasis in French Polynesia: age-specific patterns of microfilaremia, circulating antigen, and specific IgG and IgG4 responses according to transmission level.

    Science.gov (United States)

    Chanteau, S; Glaziou, P; Plichart, C; Luquiaud, P; Moulia-Pelat, J P; N'Guyen, L; Cartel, J L

    1995-01-01

    The age-specific patterns of microfilaremia, Og4C3 antigenemia, anti-Brugia malayi IgG and IgG4 were assessed in 3 villages of low, medium and high transmission level for Wuchereria bancrofti filariasis. The prevalence rates for each of the 4 markers were clearly age dependent and their patterns strongly associated with the transmission level. The antigenemia prevalence rate was consistently higher than the microfilaremia prevalence rate, in all age groups. The prevalences of anti-B. malayi IgG and IgG4 responses were very similar and much higher than those of microfilaremia or antigenemia. Antibody responses reached the plateau at an earlier age and at a higher prevalence with increased intensity of transmission. For all the markers, the prevalence rates were significantly higher in males than in females.

  18. HUMAN PARASITE SURVEY ON NASI AND BERAS ISLANDS ACEH PROVINCE, SUMATRA

    Directory of Open Access Journals (Sweden)

    E. E. Stafford

    2012-09-01

    Full Text Available Survey parasit usus dan darah manusia terhadap penduduk pulau-pulau Nasi/Beras Propinsi Aceh, Sumatra, telah diadakan dihulan Januari, 1975. Sebanyak 83 pulasan darah dari 67 pria dan 16 wanita, serta 87 contoh tinja diperoleh dari 52 pria dan 35 wanita. Brugia malayi microfilaria ditemukan dalam 3 atau 3 persen dari darah yang diperiksa dan juga parasitemia yang disebabkan oleh Plasmodium malariae 1 atau 1 persen dan P. falciparum 2 atau 2 persen. Trichuris trichiura (86 persen , merupakan parasit usus yang paling banyak ditemukan, diikuti oleh cacing tambang (77 persen, Ascaris lumbricoides (60 persen, Entamoeba histolyrica (11 per sen, H. coli (10 persen . Endolimax nana hanya 5 atau 6 persen dan Iodamoeba butschlii dan Giardia lamblia, masing-masing 3 persen. Tidak ada ditemukan Schistosoma japonicum atau pun ova cestoda diantara penduduk yang diperiksa.

  19. Parasitic diseases of the pleura.

    Science.gov (United States)

    Lal, Chitra; Huggins, John Terrill; Sahn, Steven A

    2013-05-01

    Parasitic infections are prevalent in certain parts of the world and may cause pleural involvement, which often goes unrecognized. Common parasites involving the pleura include Entamoeba histolytica, Echinococcus granulosus and Paragonimus westermani. Amebiasis can cause empyema with "anchovy sauce" pus, reactive pleural effusions and bronchopleural fistula with hydropneumothorax. Echinococcosis may result in pleural thickening, pneumothorax, secondary pleural hydatidosis and pleural effusions. Paragonimiasis may cause chylous and cholesterol pleural effusions, pleural thickening and pneumothorax. Less commonly, pulmonary eosinophilia, or Loeffler's syndrome, caused by Ascaris lumbricoides, Ancylostoma duodenale and Necator americanus and tropical pulmonary eosinophilia caused by Wuchereria bancrofti and Brugia malayi may involve the pleura. This article provides a comprehensive review of parasitic infections involving the pleura. A high index of suspicion in the appropriate clinical setting is required to facilitate prompt diagnosis and treatment of these diseases.

  20. Plasmodium knowlesi and Wucheriria bancrofti: Their vectors and challenges for the future

    Directory of Open Access Journals (Sweden)

    Indra eVythilingam

    2012-05-01

    Full Text Available Malaria and filariasis still continue to pose public health problems in developing countries of the tropics. Although plans are ongoing for the elimination of both these parasitic vector borne diseases, we are now faced with a daunting challenge as we have a fifth species, Plasmodium knowlesi a simian malaria parasite affecting humans. Similarly in peninsular Malaysia, filariasis was mainly due to Brugia malayi, however, we now see cases of W. bancrofti in immigrant workers coming into the country. Work is on going to eliminate malaria and filariasis from the country. In order to be successful we need to revamp our control measures. Thus this paper attempts to review the vectors of malaria and filariasis in Southeast Asia with special emphasis on P. knowlesi and W. bancrofti and their control strategies.

  1. Presence of Wolbachia endosymbionts in microfilariae of Wuchereria bancrofti (Spirurida: Onchocercidae from different geographical regions in India

    Directory of Open Access Journals (Sweden)

    Hoti SL

    2003-01-01

    Full Text Available In view of the recent discovery of rickettsial endosymbionts, Wolbachia in lymphatic filarial parasites, Wuchereria bancrofti and Brugia malayi and subsequently of their vital role in the survival and development of the latter, antibiotics such as tetracycline are being suggested for the treatment of lymphatic filariasis, by way of eliminating the endosymbiont. But, it is essential to assess their presence in parasites from areas endemic for lymphatic filariasis before such a new control tool is employed. In the present communication, we report the detection of Wolbachia endosymbionts in microfilariae of W. bancrofti parasites collected from geographically distant locations of India, such as Pondicherry (Union Territory, Calicut (Kerala, Jagadalpur (Madhya Pradesh, Thirukoilur (TamilNadu, Chinnanergunam (TamilNadu, Rajahmundry (Andhra Pradesh, and Varanasi (Uttar Pradesh, using Wolbachia specific 16S rDNA polymerase chain reaction.

  2. Presence and gradual disappearance of filaria-specific urinary IgG4 in babies born to antibody-positive mothers: a 2-year follow-up study.

    Science.gov (United States)

    Weerasooriya, Mirani V; Itoh, Makoto; Islam, Mohammad Z; Aoki, Yoshiki; Samarawickrema, Wilfred A; Kimura, Eisaku

    2008-09-01

    A total of 14 Sri Lankan pregnant women, who were anti-Brugia pahangi urinary IgG4 positive, and their 14 newborn babies were followed up for the urinary antibody for 2 years by enzyme-linked immunosorbent assay. Eight babies showed positive IgG4 reaction, at least once within 4 months after birth. Urinary antibody titers of mothers and their babies measured around the perinatal period showed a significant positive correlation, suggesting that baby's IgG4 was transferred from the mother through the placenta. The IgG4 decreased gradually and became negative in all positive babies by day 339.3 after birth. The present result provides a basis to judge if a positive urine ELISA test among babies is due to a new filarial infection.

  3. Dicty_cDB: VFC556 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 1 AZ534763 |AZ534763.1 ENTCF77TF Entamoeba histolytica Sheared DNA Entamoeba histolytica genomic, DNA seque...ared DNA Entamoeba histolytica genomic, DNA sequence. 40 8.0 1 AQ947500 |AQ947500.1 Sheared DNA-39L12.TF She...ared DNA Trypanosoma brucei genomic clone Sheared DNA-39...rse sequence. 40 8.0 1 AZ551370 |AZ551370.1 ENTEK19TR Entamoeba histolytica Sheared DNA Entamoeba histolytic...SAW96MLW-BmAF) Brugia malayi cDNA clone AFCW8C11 5', mRNA sequence. 40 8.0 1 BH161626 |BH161626.1 ENTSA17TF Entamoeba histolytica She

  4. A PRELIMINARY STUDY OF MALAYAN FILARIASIS IN PUDING VILLAGE, JAMBI PROVINCE (SUMATERA, INDONESIA

    Directory of Open Access Journals (Sweden)

    Sudomo M.

    2012-09-01

    Full Text Available Beberapa daerah di Propinsi Jambi akan dikembangkan menjadi daerah transmigrasi, satu di antara­nya adalah daerah Kumpeh yang terletak berdekatan dengan daerah endemik filariasis malayi. Desa yang paling dekat dengan lokasi transmigrasi tersebut adalah desa Puding. Penelitian pendahuluan tentang penyakit filariasis telah dikerjakan di desa Puding untuk mengetahui tingkat endemisitas, periodisitas B. malayi, fauna nyamuk, jenis nyamuk yang potensial menjadi vektor filariasis, hospes reservoir dan keadaan sosial-ekonomi-budaya penduduk setempat. Mf rate pada penduduk desa Puding adalah 18,7% dan dari B. malayi jenis subperiodiknokturna. Nyamuk yang tertangkap terdiri dari enam genera yaitu genus Anopheles, Aedes, Culex, Coquilletidia, Mansonia dan Tripteroides. Dari enam genera tersebut yang potensial untuk menjadi vektor filariasis adalah genus Mansonia dan ini didukung dengan diketemukannyd larva stadium L3 (infektif Brugia sp di tubuh nyamuk tersebut. Keadaan sosial-ekonomi-budaya, khususnya menyangkut adat istiadat dan kebiasaan penduduk setempat, telah dipelajari.

  5. Cystatin F Ensures Eosinophil Survival by Regulating Granule Biogenesis.

    Science.gov (United States)

    Matthews, Stephen P; McMillan, Sarah J; Colbert, Jeff D; Lawrence, Rachel A; Watts, Colin

    2016-04-19

    Eosinophils are now recognized as multifunctional leukocytes that provide critical homeostatic signals to maintain other immune cells and aid tissue repair. Paradoxically, eosinophils also express an armory of granule-localized toxins and hydrolases believed to contribute to pathology in inflammatory disease. How eosinophils deliver their supporting functions while avoiding self-inflicted injury is poorly understood. We have demonstrated that cystatin F (CF) is a critical survival factor for eosinophils. Eosinophils from CF null mice had reduced lifespan, reduced granularity, and disturbed granule morphology. In vitro, cysteine protease inhibitors restored granularity, demonstrating that control of cysteine protease activity by CF is critical for normal eosinophil development. CF null mice showed reduced pulmonary pathology in a model of allergic lung inflammation but also reduced ability to combat infection by the nematode Brugia malayi. These data identify CF as a "cytoprotectant" that promotes eosinophil survival and function by ensuring granule integrity. VIDEO ABSTRACT.

  6. STUDI ENDEMISITAS FILARIASIS DI WILAYAH KECAMATAN PEMAYUNG, KABUPATEN BATANGHARI PASCA PENGOBATAN MASSAL TAHAP III

    Directory of Open Access Journals (Sweden)

    Yahya Yahya

    2013-05-01

    Full Text Available Abstract Filariasis endemicity research in District Pemayung, Batanghari Regency Post-Mass Drug Administration Phase III has been implemented. The study aims to determine the prevalence of filariasis, microfilaria worm species, the periodicity, reservoir determination and evaluate the results of mass treatment activities that have been 3 times. The number of people who checked their blood preparation for the examination as many as 538. Blood sampling for the periodicity of the parasite examinations performed on 4 persons, each carried out blood sampling every 2 hours for 24 hours. People microfilariae with microfilariae positive number as many as 8 people to rate microfilariae (Mf rate 1.5%.. The highest parasite density of 17.493 per 20 cu mm of blood occurred at 1:00 am and decresing to 0,415 per 20 cu mm of blood at 07.00 am. The parasite was found in sub periodic nokturna 3 subjects and 1 subject was found only be found in the morning and afternoon. The results of examination of 12 cats and two monkeys were found two positive cats with Brugia malayi microfilariae. Cats that were examined and the positive was one house cat and one stray cat. The conclusion from this study showed that filariasis was still endemic with periodicity of microfilariae was sub periodic nokturna and was zoonotic. Recommendations of this study was that mass treatment  was done by giving the drug directly and took medicine in front of the officers, examination and treatment of microfilariae positive cats. Key words: microfilariae rate, periodicity, Brugia malayi, reservoir. Abstrak  Submit : 28-03-2012  Review : 04-04-2012 Review : 11-06-2012 revisi : 29–08-2012Penelitian untuk menentukan tingkat endemisitas filariasis di wilayah Kecamatan Pemayung, Kabupaten Batanghari Pasca Pengobatan Massal Tahap III telah dilaksanakan. Penelitian bertujuan untuk mengetahui prevalensi filariasis, mengetahui spesies cacing mikrofilaria, periodisitas mikrofilaria dan pemeriksaan

  7. Interdomain lateral gene transfer of an essential ferrochelatase gene in human parasitic nematodes.

    Science.gov (United States)

    Wu, Bo; Novelli, Jacopo; Jiang, Daojun; Dailey, Harry A; Landmann, Frédéric; Ford, Louise; Taylor, Mark J; Carlow, Clotilde K S; Kumar, Sanjay; Foster, Jeremy M; Slatko, Barton E

    2013-05-01

    Lateral gene transfer events between bacteria and animals highlight an avenue for evolutionary genomic loss/gain of function. Herein, we report functional lateral gene transfer in animal parasitic nematodes. Members of the Nematoda are heme auxotrophs, lacking the ability to synthesize heme; however, the human filarial parasite Brugia malayi has acquired a bacterial gene encoding ferrochelatase (BmFeCH), the terminal step in heme biosynthesis. BmFeCH, encoded by a 9-exon gene, is a mitochondrial-targeted, functional ferrochelatase based on enzyme assays, complementation, and inhibitor studies. Homologs have been identified in several filariae and a nonfilarial nematode. RNAi and ex vivo inhibitor experiments indicate that BmFeCH is essential for viability, validating it as a potential target for filariasis control.

  8. Evidence for Wolbachia symbiosis in microfilariae of Wuchereria bancrofti from West Bengal, India

    Indian Academy of Sciences (India)

    Prajna Gayen; Sudipta Maitra; Sutapa Datta; Santi P Sinha Babu

    2010-03-01

    Wolbachia are symbiotic endobacteria that infect the majority of filarial nematodes, including Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus. Recent studies have suggested that Wolbachia are necessary for the reproduction and survival of filarial nematodes and have highlighted the use of antibiotic therapy such as tetracycline/doxycycline as a novel method of treatment for infections caused by these organisms. Before such therapy is conceived and implemented on a large scale, it is necessary to assess the prevalence of the endosymbiont in W. bancrofti from different geographical locations. We present data from molecular and electron microscopic studies to provide evidence for Wolbachia symbiosis in W. bancrofti microfilariae collected from two districts (Bankura and Birbhum) of West Bengal, India.

  9. FAKTOR-FAKTOR YANG MEMPENGARUHI KEJADIAN FILARIASIS DI INDONESIA (DATA RISKESDAS 2007

    Directory of Open Access Journals (Sweden)

    Mardiana Mardiana

    2012-11-01

    Full Text Available Filariasis or elephantiasis diseases which caused by filaria worm and contagious through mosquito bite, still the major community health problem in Indonesia. There are several type of filaria worm in Indonesia, i.e. Wuchereria bancrofti, Brugia malayi and Brugia timori. The vectors of filariasis are Culex quinquefasciatus in the urban area, Anopheles spp, Aedes spp and Mansonia spp in the rural area. The infection risk in some area of filariasis related to the situation of local area. Various factor of environmental area which area physical, biological and also cultural social to be influence to development of transmitted filariasis by mosquito. The analysis of data Riskesdas 2007 has been done to perform of factor influence filariasis case in Indonesia. Same parameters was analyzed to case of filariasis in last 12 months; gender, ages, educations, work, mosquito net usage, sources of water, effluent dismissal, residences, water dismissal channel, existence of livestock in house. From analysis inferential, show there is no relation between genders, age, education, work, and mosquito net usage, sources of water, water dismissal channel, and existence of livestock in house to case filariasis. Statistically indicates that there is significantly difference between residences in rural and in urban to case of filariasis in last 12 months. Responder who live in rural areas (0,05% have 2,4 times risk higher than responder who live in urban (0,03%. The same as condition of water dismissal channel shows to existence of significantly differences. Responder who have water dismissal channel without cover have high risk infections of filariasis in the last 12 months were 0,05%, while the responder have water dismissal channel with cover have high risk in last 12 months were 0,03%. Keywords: Filariasis, endemic area, factors

  10. Mosquito infection responses to developing filarial worms.

    Directory of Open Access Journals (Sweden)

    Sara M Erickson

    Full Text Available Human lymphatic filariasis is a mosquito-vectored disease caused by the nematode parasites Wuchereria bancrofti, Brugia malayi and Brugia timori. These are relatively large roundworms that can cause considerable damage in compatible mosquito vectors. In order to assess how mosquitoes respond to infection in compatible mosquito-filarial worm associations, microarray analysis was used to evaluate transcriptome changes in Aedes aegypti at various times during B. malayi development. Changes in transcript abundance in response to the different stages of B. malayi infection were diverse. At the early stages of midgut and thoracic muscle cell penetration, a greater number of genes were repressed compared to those that were induced (20 vs. 8. The non-feeding, intracellular first-stage larvae elicited few differences, with 4 transcripts showing an increased and 9 a decreased abundance relative to controls. Several cecropin transcripts increased in abundance after parasites molted to second-stage larvae. However, the greatest number of transcripts changed in abundance after larvae molted to third-stage larvae and migrated to the head and proboscis (120 induced, 38 repressed, including a large number of putative, immunity-related genes (approximately 13% of genes with predicted functions. To test whether the innate immune system of mosquitoes was capable of modulating permissiveness to the parasite, we activated the Toll and Imd pathway controlled rel family transcription factors Rel1 and Rel2 (by RNA interference knockdown of the pathway's negative regulators Cactus and Caspar during the early stages of infection with B. malayi. The activation of either of these immune signaling pathways, or knockdown of the Toll pathway, did not affect B. malayi in Ae. aegypti. The possibility of LF parasites evading mosquito immune responses during successful development is discussed.

  11. Filarial worms reduce Plasmodium infectivity in mosquitoes.

    Directory of Open Access Journals (Sweden)

    Matthew T Aliota

    Full Text Available BACKGROUND: Co-occurrence of malaria and filarial worm parasites has been reported, but little is known about the interaction between filarial worm and malaria parasites with the same Anopheles vector. Herein, we present data evaluating the interaction between Wuchereria bancrofti and Anopheles punctulatus in Papua New Guinea (PNG. Our field studies in PNG demonstrated that An. punctulatus utilizes the melanization immune response as a natural mechanism of filarial worm resistance against invading W. bancrofti microfilariae. We then conducted laboratory studies utilizing the mosquitoes Armigeres subalbatus and Aedes aegypti and the parasites Brugia malayi, Brugia pahangi, Dirofilaria immitis, and Plasmodium gallinaceum to evaluate the hypothesis that immune activation and/or development by filarial worms negatively impact Plasmodium development in co-infected mosquitoes. Ar. subalbatus used in this study are natural vectors of P. gallinaceum and B. pahangi and they are naturally refractory to B. malayi (melanization-based refractoriness. METHODOLOGY/PRINCIPAL FINDINGS: Mosquitoes were dissected and Plasmodium development was analyzed six days after blood feeding on either P. gallinaceum alone or after taking a bloodmeal containing both P. gallinaceum and B. malayi or a bloodmeal containing both P. gallinaceum and B. pahangi. There was a significant reduction in the prevalence and mean intensity of Plasmodium infections in two species of mosquito that had dual infections as compared to those mosquitoes that were infected with Plasmodium alone, and was independent of whether the mosquito had a melanization immune response to the filarial worm or not. However, there was no reduction in Plasmodium development when filarial worms were present in the bloodmeal (D. immitis but midgut penetration was absent, suggesting that factors associated with penetration of the midgut by filarial worms likely are responsible for the observed reduction in malaria

  12. Evaluation of immune response elicited by inulin as an adjuvant with filarial antigens in mice model.

    Science.gov (United States)

    Mahalakshmi, N; Aparnaa, R; Kaliraj, P

    2014-10-01

    Filariasis caused by infectious parasitic nematodes has been identified as the second leading source of permanent and long-term disability in Sub-Saharan Africa, Asia and Latin America. Several vaccine candidates were identified from infective third-stage larvae (L3) which involves in the critical transition from arthropod to human. Hitherto studies of these antigens in combination with alum adjuvant have shown to elicit its characteristic Th2 responses. Inulin is a safe, non-toxic adjuvant that principally stimulates the innate immune response through the alternative complement pathway. In the present study, the immune response elicited by inulin and alum as adjuvants were compared with filarial antigens from different aetiological agents: secreted larval acidic protein 1 (SLAP1) from Onchocerca volvulus and venom allergen homologue (VAH) from Brugia malayi as single or as cocktail vaccines in mice model. The study revealed that inulin can induce better humoral response against these antigens than alum adjuvant. Antibody isotyping disclosed inulin's ability to elevate the levels of IgG2a and IgG3 antibodies which mediates in complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC), respectively, in mice. Splenocyte analysis showed that T cells prestimulated with inulin have higher stimulation index (P inulin formulation had induced higher cytotoxicity with filarial antigens (as single P inulin to deplete the levels of Treg and brought a balance in Th1/Th2 arms against filarial antigens in mice.

  13. Rendering the Intractable More Tractable: Tools from Caenorhabditis elegans Ripe for Import into Parasitic Nematodes.

    Science.gov (United States)

    Ward, Jordan D

    2015-12-01

    Recent and rapid advances in genetic and molecular tools have brought spectacular tractability to Caenorhabditis elegans, a model that was initially prized because of its simple design and ease of imaging. C. elegans has long been a powerful model in biomedical research, and tools such as RNAi and the CRISPR/Cas9 system allow facile knockdown of genes and genome editing, respectively. These developments have created an additional opportunity to tackle one of the most debilitating burdens on global health and food security: parasitic nematodes. I review how development of nonparasitic nematodes as genetic models informs efforts to import tools into parasitic nematodes. Current tools in three commonly studied parasites (Strongyloides spp., Brugia malayi, and Ascaris suum) are described, as are tools from C. elegans that are ripe for adaptation and the benefits and barriers to doing so. These tools will enable dissection of a huge array of questions that have been all but completely impenetrable to date, allowing investigation into host-parasite and parasite-vector interactions, and the genetic basis of parasitism.

  14. [Scanning microscopical observations on the foregut structures o mosquitoes and their role for the ingestion of microfilariae (author's transl)].

    Science.gov (United States)

    Buse, E; Kuhlow, F

    1979-12-01

    Experiments on the transmission of Brugia malayi by various mosquitoes had shown that microfilariae ingested by some species were badly damaged when they reached the stomach, but were much less hurt in others. The structures of the foregut likely to cause these injuries, were investigated and documented by scanning microscope techniques. In Anopheles albimanus, A. arabiensis, A. stephensi and A. pharoensis which have well developed armatures the microfilariae showed a high rate of destruction. In A. stroparvus as well as in Aedes aegypti, Ae. togoi and Culex fatigans in which these structures are missing or poorly developed the larvae were much less affected. From the size, shape and position of the different papillae, spines, rods and cones observed it can be concluded and confirmed that the pharyngeal armature (buccopharyngeal bar) will be by far the most important structure responsible for the injuries of the microfilariae. However, it appears that the characteristics of different filaria species can play an important role in preventing such damages.

  15. Genome mining offers a new starting point for parasitology research.

    Science.gov (United States)

    Lv, Zhiyue; Wu, Zhongdao; Zhang, Limei; Ji, Pengyu; Cai, Yifeng; Luo, Shiqi; Wang, Hongxi; Li, Hao

    2015-02-01

    Parasites including helminthes, protozoa, and medical arthropod vectors are a major cause of global infectious diseases, affecting one-sixth of the world's population, which are responsible for enormous levels of morbidity and mortality important and remain impediments to economic development especially in tropical countries. Prevalent drug resistance, lack of highly effective and practical vaccines, as well as specific and sensitive diagnostic markers are proving to be challenging problems in parasitic disease control in most parts of the world. The impressive progress recently made in genome-wide analysis of parasites of medical importance, including trematodes of Clonorchis sinensis, Opisthorchis viverrini, Schistosoma haematobium, S. japonicum, and S. mansoni; nematodes of Brugia malayi, Loa loa, Necator americanus, Trichinella spiralis, and Trichuris suis; cestodes of Echinococcus granulosus, E. multilocularis, and Taenia solium; protozoa of Babesia bovis, B. microti, Cryptosporidium hominis, Eimeria falciformis, E. histolytica, Giardia intestinalis, Leishmania braziliensis, L. donovani, L. major, Plasmodium falciparum, P. vivax, Trichomonas vaginalis, Trypanosoma brucei and T. cruzi; and medical arthropod vectors of Aedes aegypti, Anopheles darlingi, A. sinensis, and Culex quinquefasciatus, have been systematically covered in this review for a comprehensive understanding of the genetic information contained in nuclear, mitochondrial, kinetoplast, plastid, or endosymbiotic bacterial genomes of parasites, further valuable insight into parasite-host interactions and development of promising novel drug and vaccine candidates and preferable diagnostic tools, thereby underpinning the prevention and control of parasitic diseases.

  16. Restriction fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the mosquito Aedes aegypti

    Energy Technology Data Exchange (ETDEWEB)

    Severson, D.W.; Thathy, V.; Mori, A. [Univ. of Wisconsin, Madison, WI (United States)] [and others

    1995-04-01

    Susceptibility of the mosquito Aedes aegypti to the malarial parasite Plasmodium gallinaceum was investigated as a quantitative trait using restriction fragment length polymorphisms (RFLP). Two F{sub 2} populations of mosquitoes were independently prepared from pairwise matings between a highly susceptible and a refractory strain of A. aegypti. RFLP were tested for association with oocyst development on the mosquito midgut. Two putative quantitative trait loci (QTL) were identified that significantly affect susceptibility. One QTL, pgs [2,LF98], is located on chromosome 2 and accounted for 65 and 49% of the observed phenotypic variance in the two populations, respectively. A second QTL, pgs[3,MalI], is located on chromosome 3 and accounted for 14 and 10% of the observed phenotypic variance in the two populations, respectively. Both QTL exhibit a partial dominance effect on susceptibility, wherein the dominance effect is derived from the refractory parent. No indication of epistasis between these QTL was detected. Evidence suggests that either a tightly linked cluster of independent genes or a single locus affecting susceptibility to various mosquito-borne parasites and pathogens has evolved near the LF98 locus; in addition to P. gallinaceum susceptibility, this general genome region has previously been implicated in susceptibility to the filaria nematode Brugia malayi and the yellow fever virus. 35 refs., 2 figs., 3 tabs.

  17. Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence

    Science.gov (United States)

    Gordon, Kacy L.; Arthur, Robert K.; Ruvinsky, Ilya

    2015-01-01

    Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements. PMID:26020930

  18. Antifilarial Lead Molecules Isolated from Trachyspermum ammi

    Directory of Open Access Journals (Sweden)

    Kalyanasundaram Muthuswamy

    2008-09-01

    Full Text Available Lymphatic filariasis is caused by infection with the parasitic filarial nematodes Wuchereria bancrofti, Brugia malayi and B. timori, transmitted by mosquitoes. The lack of an adulticidal drug poses a challenge to filariasis elimination, hence it is essential to develop an effective antifilarial drug which could either kill or permanently sterilize the adult worms. In the reported work the in vitro activity of a methanolic extract of fruits of Trachyspermum ammi (Apiaceae against adult bovine filarial Setaria digitata worms has been investigated. A bioassay-guided fractionation was carried out by subjecting the crude extract to flash chromatography. HPLC analysis was done for the crude extract and active fraction. The crude extract and the active fraction showed significant activity against the adult S. digitata by both a worm motility and MTT [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] reduction assays. The isolated active principle was chemically characterized by IR, 1H-NMR and MS analysis and identified as a phenolic monoterpene. It was screened for in vivo antifilarial activity against the human filarial worm B. malayi in Mastomys coucha, showing macrofilaricidal activity and female worm sterility in vivo against B. malayi. The findings thus provide a new lead for development of a macrofilaricidal drug from natural products

  19. Review of zoonotic parasites in medical and veterinary fields in the Republic of Korea.

    Science.gov (United States)

    Youn, Heejeong

    2009-10-01

    Zoonotic parasites are animal parasites that can infect humans. The major zoonotic protozoa in the Republic of Korea are Babesia bovis, Chilomastix mesnili, Cryptosporidium parvum, Endolimax nana, Entamoeba coli, Entamoeba hitolytica, Giardia lamblia, Iodamoeba bütschlii, Pneumocystis carinii, Sarcocystis cruzi, and Toxoplasma gondii. The major zoonotic helminths in Korea include trematodes, cestodes, and nematodes. Trematodes are Clonorchis sinensis, Echinostoma hortense, Echinostoma spp., Fasciola hepatica, Heterophyes nocens, Metagonimus yokogawai, and Paragonimus westermani. Cestodes are Diphyllobothrium latum, Dipylidium caninum, Echinococcus granulosus, Hymenolepis nana, Raillietina tetragona, sparganum (Spirometra spp.), Taenia saginata, T. solium, and T. asiatica. Nematodes are Ancylostoma caninum, Brugia malayi, Capillaria hepatica, Dirofilaria immitis, Gnathostoma dololesi, Gnathostoma spinigerum, Loa loa, Onchocerca gibsoni, Strongyloides stercoralis, Thelazia callipaeda, Trichinella spiralis, Trichostrongylus orientalis, Trichuris trichiura, and Trichuris vulpis. The one arthropod is Sarcoptes scabiei. Many of these parasites have disappeared or were in decline after the 1990's. Since the late 1990's, the important zoonotic protozoa have been C. parvum, E. nana, E. coli, E. hitolytica, G. lamblia, I. buetschlii, P. carinii and T. gondii. The important zoonotic helminths have been C. sinensis, H. nocens, M. yokogawai, P. westermani, D. latum, T. asiatica, sparganum, B. malayi, T. orientalis, T. callipaeda and T. spiralis. However, outbreaks of these parasites are only in a few endemic areas. The outbreaks of Enterobius vermicularis and head lice, human parasites, have recently increased in the kindergartens and primary schools in the Republic of Korea.

  20. Open source tool for prediction of genome wide protein-protein interaction network based on ortholog information

    Directory of Open Access Journals (Sweden)

    Pedamallu Chandra Sekhar

    2010-08-01

    Full Text Available Abstract Background Protein-protein interactions are crucially important for cellular processes. Knowledge of these interactions improves the understanding of cell cycle, metabolism, signaling, transport, and secretion. Information about interactions can hint at molecular causes of diseases, and can provide clues for new therapeutic approaches. Several (usually expensive and time consuming experimental methods can probe protein - protein interactions. Data sets, derived from such experiments make the development of prediction methods feasible, and make the creation of protein-protein interaction network predicting tools possible. Methods Here we report the development of a simple open source program module (OpenPPI_predictor that can generate a putative protein-protein interaction network for target genomes. This tool uses the orthologous interactome network data from a related, experimentally studied organism. Results Results from our predictions can be visualized using the Cytoscape visualization software, and can be piped to downstream processing algorithms. We have employed our program to predict protein-protein interaction network for the human parasite roundworm Brugia malayi, using interactome data from the free living nematode Caenorhabditis elegans. Availability The OpenPPI_predictor source code is available from http://tools.neb.com/~posfai/.

  1. Targeting the Wolbachia cell division protein FtsZ as a new approach for antifilarial therapy.

    Directory of Open Access Journals (Sweden)

    Zhiru Li

    2011-11-01

    Full Text Available The use of antibiotics targeting the obligate bacterial endosymbiont Wolbachia of filarial parasites has been validated as an approach for controlling filarial infection in animals and humans. Availability of genomic sequences for the Wolbachia (wBm present in the human filarial parasite Brugia malayi has enabled genome-wide searching for new potential drug targets. In the present study, we investigated the cell division machinery of wBm and determined that it possesses the essential cell division gene ftsZ which was expressed in all developmental stages of B. malayi examined. FtsZ is a GTPase thereby making the protein an attractive Wolbachia drug target. We described the molecular characterization and catalytic properties of Wolbachia FtsZ. We also demonstrated that the GTPase activity was inhibited by the natural product, berberine, and small molecule inhibitors identified from a high-throughput screen. Furthermore, berberine was also effective in reducing motility and reproduction in B. malayi parasites in vitro. Our results should facilitate the discovery of selective inhibitors of FtsZ as a novel anti-symbiotic approach for controlling filarial infection. NOTE: The nucleotide sequences reported in this paper are available in GenBank™ Data Bank under the accession number wAlB-FtsZ (JN616286.

  2. Interleukin-12 suppresses filaria-induced pulmonary eosinophilia, deposition of major basic protein and airway hyperresponsiveness.

    Science.gov (United States)

    Mehlotra, R K; Hall, L R; Higgins, A W; Dreshaj, I A; Haxhiu, M A; Kazura, J W; Pearlman, E

    1998-10-01

    Tropical Pulmonary Eosinophilia (TPE) is a severe form of allergic asthma caused by the host inflammatory response to filarial helminths in the lung microvasculature, and is characterized by pulmonary eosinophilia, increased filarial-specific IgG and IgE antibodies, and airway hyperresponsiveness. The current study examined the effect of IL-12 on pulmonary eosinophilia, deposition of eosinophil major basic protein and airway hyperresponsiveness in mice inoculated i.v. with Brugia malayi microfilariae. Injection of recombinant murine IL-12 modulated the T helper (Th) response in the lungs from Th2- to Th1-like, with elevated IFN-gamma, and decreased IL-4 and IL-5 production. Consistent with this shift in cytokine response, antigen-specific IgG2a was elevated, and IgG1 and total serum IgE were decreased. In addition, eosinophils in BAL fluid from IL-12 treated mice were reduced from 56% to 11%, and there was no detectable MBP on respiratory epithelial cells. Importantly, IL-12 suppressed airway hyperresponsiveness compared with saline-injected control animals. Taken together, these data clearly demonstrate that by modulating Th associated cytokine production, IL-12 down-regulates filaria-induced lung immunopathology.

  3. A high resolution melting real time PCR for mapping of filaria infection in domestic cats living in brugian filariosis-endemic areas.

    Science.gov (United States)

    Wongkamchai, Sirichit; Nochote, Hathai; Foongladda, Suporn; Dekumyoy, Paron; Thammapalo, Suvit; Boitano, John J; Choochote, Wej

    2014-03-17

    We present here a real time PCR with high resolution melting (HRM) analysis for determining the prevalence and distribution of filarial species in domestic cats residing in brugian filariosis endemic areas of Narathiwat province, Thailand. Filarial species can be clearly distinguished in a single well using a single pair of primers. Blood samples were taken from a total of 2039 domestic cats living in endemic areas. Microfilariae were detected in 5.7% of the sample, while the overall prevalence of filaria infection by HRM analysis was 6.6%. The filariae species found in the infected cats were Brugia malayi, Dirofilaria immitis, D. repens as well as Acanthocheilonema (Dipetalonema) reconditum. This is the first report of A. reconditum infection from Thailand. The study also observed an overlapping of the distribution areas of animal and human filariae. From a public health perspective, the distribution and prevalence of these nematodes warrant an appropriate drug-based prophylaxis to be administered to cats in the endemic areas to reduce the number of diseased carriers. Furthermore, this molecular approach is more sensitive than microfilariae detection, enables species identification and greatly facilitates the collection of epidemiological data. Thus, the present study may help to bridge human-animal interface by coordinating research outcomes with the control of zoonoses that is vitally important for human and veterinary public health.

  4. Regulatory T cells in human lymphatic filariasis: stronger functional activity in microfilaremics.

    Directory of Open Access Journals (Sweden)

    Linda J Wammes

    Full Text Available Infection with filarial parasites is associated with T cell hyporesponsiveness, which is thought to be partly mediated by their ability to induce regulatory T cells (Tregs during human infections. This study investigates the functional capacity of Tregs from different groups of filarial patients to suppress filaria-specific immune responses during human filariasis. Microfilaremic (MF, chronic pathology (CP and uninfected endemic normal (EN individuals were selected in an area endemic for Brugia timori in Flores island, Indonesia. PBMC were isolated, CD4CD25(hi cells were magnetically depleted and in vitro cytokine production and proliferation in response to B. malayi adult worm antigen (BmA were determined in total and Treg-depleted PBMC. In MF subjects BmA-specific T and B lymphocyte proliferation as well as IFN-gamma, IL-13 and IL-17 responses were lower compared to EN and CP groups. Depletion of Tregs restored T cell as well as B cell proliferation in MF-positives, while proliferative responses in the other groups were not enhanced. BmA-induced IL-13 production was increased after Treg removal in MF-positives only. Thus, filaria-associated Tregs were demonstrated to be functional in suppressing proliferation and possibly Th2 cytokine responses to BmA. These suppressive effects were only observed in the MF group and not in EN or CP. These findings may be important when considering strategies for filarial treatment and the targeted prevention of filaria-induced lymphedema.

  5. MENGENAL PARASIT FILARIA

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    Tri Ramadhani

    2012-11-01

    Full Text Available Filariasis atau kaki gajah adalah penyakit menular yang disebabkan karena infeksi cacing filaria yang hidup disaluran dan kelenjar getah bening (limfe serta menyebabkan gejala akut, kronis. Filariasis mulai dikenal di Indonesia tahun 1889 sejak Haga dan Van Eecke menemukan kasus pembesaran scrotum di Jakarta. Penyakit tersebut dapat menular kepada orang lain dengan perantara gigitan nyamuk. Seluruh wilayah Indonesia berpotensi untuk terjangkitnya penyakit tersebut, hal ini mengingat cacing sebagai penyebabnya dan nyamuk penularnya tersebar luas. Keadaan ini didukung oleh kerusakan lingkungan, seperti banjir, penebangan hutan dan lainnya yang memperluas tempat berkembangbiaknya nyamuk. Meskipun filariasis tidak mematikan secara langsung, dengan adanya demam dan bisul-bisul (abses yang hilang timbul, dan gejala menahun berupa pembesaran/elefantiasis yang merupakan cacat menetap akan sangat mengganggu. Secara ekonomis keadaan tersebut sangat merugikan, karena mengurangi produktivitas masyarakat, serta diperlukan biaya pengobatan dan perawatan yang tidak mudah dan tidak murah.Di Indonesia filariasis limfatik di sebabkan oleh tiga spesies cacing filaria yaitu Brugia malayi,B.timori dan Wuchereria bancrofti, yang terbagi lagi menjadi 6 tipe secara epidemiologi.Tiap parasit mempunyai siklus hidup yang kompleks dan infeksi pada manusia tidak akan berhasil kecuali jika terjadi pemaparan larva infektif untuk waktu yang lama. Setelah terjadi pemaparan, dibutuhkan waktu bertahun-tahun sebelum timbulnya perubahan patologis yang nyata pada manusia. Periodisitas dalam sirkulasi setiap mikrofilaria akan berbeda, tergantung dari spesiesnya.

  6. Filaria/Wolbachia activation of dendritic cells and development of Th1-associated responses is dependent on Toll-like receptor 2 in a mouse model of ocular onchocerciasis (river blindness).

    Science.gov (United States)

    Daehnel, K; Gillette-Ferguson, I; Hise, A G; Diaconu, E; Harling, M J; Heinzel, F P; Pearlman, E

    2007-09-01

    Toll-like receptors (TLRs) regulate dendritic cell function and activate signals that mediate the nature of the adaptive immune response. The current study examined the role of TLRs in dendritic cell activation and in regulating T cell and antibody responses to antigens from the filarial parasites Onchocerca volvulus and Brugia malayi, which cause river blindness and lymphatic filariasis, respectively. Bone-marrow-derived CD11c(+) cells from C57BL/6 and TLR4(-/-) mice produced high levels of IL-6 and RANTES, and showed elevated surface CD40 expression, whereas CD11c(+) cells from myeloid differentiation factor 88(-/-) (MyD88(-/-)), TLR2(-/-) and TLR2/4(-/-) mice were not activated. Similarly, IFN-gamma production by splenocytes from immunized TLR2(-/-) mice was significantly impaired compared with splenocytes from C57BL/6 and TLR4(-/-) mice. In contrast, there was no difference among these strains in Th2-associated responses including IL-5 production by splenocytes from immunized animals, serum IgE and IgG(1), or eosinophil infiltration into the corneal stroma. Neutrophil recruitment to the cornea and CXC chemokine production was inhibited in immunized TLR2(-/-) mice compared with C57BL/6 and TLR4(-/-) mice. Taken together, these findings demonstrate an essential role for TLR2 in filaria-induced dendritic cell activation, IFN-gamma production and neutrophil migration to the cornea, but does not affect filaria-induced Th2-associated responses.

  7. Recent Advances on the Use of Biochemical Extracts as Filaricidal Agents

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    Nazeh M. Al-Abd

    2013-01-01

    Full Text Available Lymphatic filariasis is a parasitic infection that causes a devastating public health and socioeconomic burden with an estimated infection of over 120 million individuals worldwide. The infection is caused by three closely related nematode parasites, namely, Wuchereria bancrofti, Brugia malayi, and B. timori, which are transmitted to human through mosquitoes of Anopheles, Culex, and Aedes genera. The species have many ecological variants and are diversified in terms of their genetic fingerprint. The rapid spread of the disease and the genetic diversification cause the lymphatic filarial parasites to respond differently to diagnostic and therapeutic interventions. This in turn prompts the current challenge encountered in its management. Furthermore, most of the chemical medications used are characterized by adverse side effects. These complications urgently warrant intense prospecting on bio-chemicals that have potent efficacy against either the filarial worms or thier vector. In lieu of this, we presented a review on recent literature that reported the efficacy of filaricidal biochemicals and those employed as vector control agents. In addition, methods used for biochemical extraction, screening procedures, and structure of the bioactive compounds were also presented.

  8. Construction and characterization of an expressed sequenced tag library for the mosquito vector Armigeres subalbatus

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    Tsai Shih-Feng

    2007-12-01

    Full Text Available Abstract Background The mosquito, Armigeres subalbatus, mounts a distinctively robust innate immune response when infected with the nematode Brugia malayi, a causative agent of lymphatic filariasis. In order to mine the transcriptome for new insight into the cascade of events that takes place in response to infection in this mosquito, 6 cDNA libraries were generated from tissues of adult female mosquitoes subjected to immune-response activation treatments that lead to well-characterized responses, and from aging, naïve mosquitoes. Expressed sequence tags (ESTs from each library were produced, annotated, and subjected to comparative analyses. Results Six libraries were constructed and used to generate 44,940 expressed sequence tags, of which 38,079 passed quality filters to be included in the annotation project and subsequent analyses. All of these sequences were collapsed into clusters resulting in 8,020 unique sequence clusters or singletons. EST clusters were annotated and curated manually within ASAP (A Systematic Annotation Package for Community Analysis of Genomes web portal according to BLAST results from comparisons to Genbank, and the Anopheles gambiae and Drosophila melanogaster genome projects. Conclusion The resulting dataset is the first of its kind for this mosquito vector and provides a basis for future studies of mosquito vectors regarding the cascade of events that occurs in response to infection, and thereby providing insight into vector competence and innate immunity.

  9. Assembly of the Genome of the Disease Vector Aedes aegypti onto a Genetic Linkage Map Allows Mapping of Genes Affecting Disease Transmission

    KAUST Repository

    Juneja, Punita

    2014-01-30

    The mosquito Aedes aegypti transmits some of the most important human arboviruses, including dengue, yellow fever and chikungunya viruses. It has a large genome containing many repetitive sequences, which has resulted in the genome being poorly assembled - there are 4,758 scaffolds, few of which have been assigned to a chromosome. To allow the mapping of genes affecting disease transmission, we have improved the genome assembly by scoring a large number of SNPs in recombinant progeny from a cross between two strains of Ae. aegypti, and used these to generate a genetic map. This revealed a high rate of misassemblies in the current genome, where, for example, sequences from different chromosomes were found on the same scaffold. Once these were corrected, we were able to assign 60% of the genome sequence to chromosomes and approximately order the scaffolds along the chromosome. We found that there are very large regions of suppressed recombination around the centromeres, which can extend to as much as 47% of the chromosome. To illustrate the utility of this new genome assembly, we mapped a gene that makes Ae. aegypti resistant to the human parasite Brugia malayi, and generated a list of candidate genes that could be affecting the trait. © 2014 Juneja et al.

  10. A cell-based screen reveals that the albendazole metabolite, albendazole sulfone, targets Wolbachia.

    Science.gov (United States)

    Serbus, Laura R; Landmann, Frederic; Bray, Walter M; White, Pamela M; Ruybal, Jordan; Lokey, R Scott; Debec, Alain; Sullivan, William

    2012-09-01

    Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts.

  11. Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence.

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    Kacy L Gordon

    2015-05-01

    Full Text Available Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2 from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements.

  12. MID TERM ASSESSMENT OF MASS DRUG ADMINISTRATION IN LYMPHATIC FILARIASIS ENDEMIC AREA OF DAMOH AND SAGAR DISTRICT OF MADHYA PRADESH

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    Mohan

    2015-03-01

    Full Text Available BACKGROUND: Lymphatic filariasis caused by Wuchereria bancrofti and Brugia malayi is an important public health problem in India. Filariasis is a major social and the fourth most common cause of disability all over the globe. Filariasis is endemic in 17 States and six Union Territories, with about 553 million people at risk of infection. It has been a major public health problem in India. The Global Programme for Elimination of Lymphatic filariasis was launched by the WHO in 2000 with the goal of eliminating Lymphatic filariasis as a public health problem by the year 2020. For the effective control of filariasis >65% population of endemic areas should be covered by single dose of Diethylcarbamazine 6mg/kg (DEC. OBJECTIVES: To assess the coverage and compliance of mass drug administration in the selected District and to make independent assessment with respect to process and out - come indicators. MATERIAL AND METHODS : A community based cross sectional study through house to house survey method in selected clusters was adopted. An independent evaluation was done and the outcome was assessed as the coverage and compliance of mass drug administration. RESULTS: In both Damoh and Sagar Districts of Madhya Pradesh, the coverage level for DEC was > 80% in all the Blocks. CONCL USION: The mass drug administration was aimed only to distribute the drug and the issues related to compliance, proper health education and side effects management were not given enough attention. These issues are important to make programme effective.

  13. Molecular and biochemical characterization of nematode cofactor independent phosphoglycerate mutases.

    Science.gov (United States)

    Raverdy, Sylvine; Zhang, Yinhua; Foster, Jeremy; Carlow, Clotilde K S

    2007-12-01

    Phosphoglycerate mutase (PGM, EC 5.4.2.1) catalyzes the isomerization of 3-phosphoglycerate and 2-phosphoglycerate in glycolysis and gluconeogenesis. Two distinct types of PGM exist in nature, one that requires 2,3-bisphosphoglycerate as a cofactor (dPGM) and another that does not (iPGM). The two enzymes are structurally distinct and possess different mechanisms of action. In any particular organism, one form may exist or both. Nematodes possess the iPGM form whereas mammals have dPGM. In the present study, we have cloned and expressed iPGM from Onchocerca volvulus and described the catalytic properties of O. volvulus, Brugia malayi and Caenorhabditis elegans iPGM enzymes. Temperature and pH optima were determined for each enzyme. Like other iPGM enzymes, the activities of the nematode iPGM enzymes were dependent on the presence of divalent ions. Inactivation by EDTA could be restored most effectively by magnesium and manganese ions. Kinetic parameters and specific activities of the various recombinant enzymes were determined. The high similarity in catalytic properties among the enzymes indicates that a single enzyme inhibitor would likely be effective against all nematode enzymes. Inhibition of iPGM activity in vivo may lead to lethality as indicated by RNAi studies in C. elegans. Our results support the development of iPGM as a promising drug target in parasitic nematodes.

  14. Cofactor-independent phosphoglycerate mutase has an essential role in Caenorhabditis elegans and is conserved in parasitic nematodes.

    Science.gov (United States)

    Zhang, Yinhua; Foster, Jeremy M; Kumar, Sanjay; Fougere, Marjorie; Carlow, Clotilde K S

    2004-08-27

    Phosphoglycerate mutases catalyze the interconversion of 2- and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. They exist in two unrelated forms that are either cofactor (2,3-diphosphoglycerate)-dependent or cofactor-independent. The two enzymes have no similarity in amino acid sequence, tertiary structure, or catalytic mechanism. Certain organisms including vertebrates have only the cofactor-dependent form, whereas other organisms can possess the independent form or both. Caenorhabditis elegans has been predicted to have only independent phosphoglycerate mutase. In this study, we have cloned and produced recombinant, independent phosphoglycerate mutases from C. elegans and the human-parasitic nematode Brugia malayi. They are 70% identical to each other and related to known bacterial, fungal, and protozoan enzymes. The nematode enzymes possess the catalytic serine, and other key amino acids proposed for catalysis and recombinant enzymes showed typical phosphoglycerate mutase activities in both the glycolytic and gluconeogenic directions. The gene is essential in C. elegans, because the reduction of its activity by RNA interference led to embryonic lethality, larval lethality, and abnormal body morphology. Promoter reporter analysis indicated widespread expression in larval and adult C. elegans with the highest levels apparent in the nerve ring, intestine, and body wall muscles. The enzyme was found in a diverse group of nematodes representing the major clades, indicating that it is conserved throughout this phylum. Our results demonstrate that nematodes, unlike vertebrates, utilize independent phosphoglycerate mutase in glycolytic and gluconeogenic pathways and that the enzyme is probably essential for all nematodes.

  15. Targeting the Wolbachia cell division protein FtsZ as a new approach for antifilarial therapy.

    Science.gov (United States)

    Li, Zhiru; Garner, Amanda L; Gloeckner, Christian; Janda, Kim D; Carlow, Clotilde K

    2011-11-01

    The use of antibiotics targeting the obligate bacterial endosymbiont Wolbachia of filarial parasites has been validated as an approach for controlling filarial infection in animals and humans. Availability of genomic sequences for the Wolbachia (wBm) present in the human filarial parasite Brugia malayi has enabled genome-wide searching for new potential drug targets. In the present study, we investigated the cell division machinery of wBm and determined that it possesses the essential cell division gene ftsZ which was expressed in all developmental stages of B. malayi examined. FtsZ is a GTPase thereby making the protein an attractive Wolbachia drug target. We described the molecular characterization and catalytic properties of Wolbachia FtsZ. We also demonstrated that the GTPase activity was inhibited by the natural product, berberine, and small molecule inhibitors identified from a high-throughput screen. Furthermore, berberine was also effective in reducing motility and reproduction in B. malayi parasites in vitro. Our results should facilitate the discovery of selective inhibitors of FtsZ as a novel anti-symbiotic approach for controlling filarial infection. NOTE: The nucleotide sequences reported in this paper are available in GenBank™ Data Bank under the accession number wAlB-FtsZ (JN616286).

  16. Identification and characterization of the cofactor-independent phosphoglycerate mutases of Dirofilaria immitis and its Wolbachia endosymbiont.

    Science.gov (United States)

    Li, Zhiru; Galvin, Brendan D; Raverdy, Sylvine; Carlow, Clotilde K S

    2011-03-22

    Drug treatments for heartworm disease have not changed significantly in the last decade. Due to concerns about possible drug resistance and their lower efficacy against adult worms, there is a need for the development of new antifilarial drug therapies. The recent availability of genomic sequences for the related filarial parasite Brugia malayi and its Wolbachia endosymbiont enables genome-wide searching for new drug targets. Phosphoglycerate mutase (PGM) enzymes catalyze the critical isomerization of 3-phosphoglycerate (3-PG) and 2-phosphoglycerate (2-PG) in glycolytic and gluconeogenic metabolic pathways. There are two unrelated PGM enzymes, which are structurally distinct and possess different mechanisms of action. The mammalian enzyme requires 2,3-bisphosphoglycerate as a cofactor (dependent PGM or dPGM), while the other type of PGM does not (independent PGM or iPGM). In the present study, we have determined that Dirofilaria immitis and its Wolbachia endosymbiont both possess active iPGM. We describe the molecular characterization and catalytic properties of each enzyme. Our results will facilitate the discovery of selective inhibitors of these iPGMs as potentially novel drug treatments for heartworm disease.

  17. First analysis of the secretome of the canine heartworm, Dirofilaria immitis

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    Geary James

    2012-07-01

    Full Text Available Abstract Background The characterization of proteins released from filariae is an important step in addressing many of the needs in the diagnosis and treatment of these clinically important parasites, as well as contributing to a clearer understanding of their biology. This report describes findings on the proteins released during in vitro cultivation of adult Dirofilaria immitis , the causative agent of canine and feline heartworm disease. Differences in protein secretion among nematodes in vivo may relate to the ecological niche of each parasite and the pathological changes that they induce. Methods The proteins in the secretions of cultured adult worms were run on Tris-Glycine gels, bands separated and peptides from each band analysed by ultra mass spectrometry and compared with a FastA dataset of predicted tryptic peptides derived from a genome sequence of D. immitis. Results This study identified 110 proteins. Of these proteins, 52 were unique to D. immitis . A total of 23 (44% were recognized as proteins likely to be secreted. Although these proteins were unique, the motifs were conserved compared with proteins secreted by other nematodes. Conclusion The present data indicate that D. immitis secretes proteins that are unique to this species, when compared with Brugia malayi. The two major functional groups of molecules represented were those representing cellular and of metabolic processes. Unique proteins might be important for maintaining an infection in the host environment, intimately involved in the pathogenesis of disease and may also provide new tools for the diagnosis of heartworm infection.

  18. Differentially expressed genes between female and male adult Anopheles anthropophagus.

    Science.gov (United States)

    Geng, Yi-Jie; Gao, Shi-Tong; Huang, Da-Na; Zhao, Yi-Rui; Liu, Jian-ping; Li, Xiao-Heng; Zhang, Ren-Li

    2009-09-01

    The aim of the present study was to identify sex-specific genes in adult Anopheles anthropophagus. As the major malaria vector and Brugia malayi vector in the Asian continent, female Anopheles mosquitoes take blood meals and transmit pathogens through this pathway, while males are nectar feeders. This complex behavior is controlled at several levels, but is probably initiated by the genetic background difference between these two groups. In our study, a subtractive cDNA library for female A. anthropophagus was constructed using the suppression subtractive hybridization (SSH) technique and then 3,074 clones from the female SSH library were analyzed using a microarray-based survey. Genes that were expressed differentially according to sex in A. anthropophagus were screened using real-time polymerase chain reaction and reverse transcription polymerase chain reaction. In our results, we report a series of genes which may be involved in female-specific mosquito behavior, including an inorganic phosphate transporter, a serine protease, the salivary protein GP35-2, and the D7 cluster salivary protein. These findings will provide clues to the nature of insect vectors and open up unprecedented opportunities to develop novel strategies for the control of mosquito-borne diseases.

  19. The astacin metalloprotease moulting enzyme NAS-36 is required for normal cuticle ecdysis in free-living and parasitic nematodes.

    Science.gov (United States)

    Stepek, Gillian; McCormack, Gillian; Birnie, Andrew J; Page, Antony P

    2011-02-01

    Nematodes represent one of the most abundant and species-rich groups of animals on the planet, with parasitic species causing chronic, debilitating infections in both livestock and humans worldwide. The prevalence and success of the nematodes is a direct consequence of the exceptionally protective properties of their cuticle. The synthesis of this cuticle is a complex multi-step process, which is repeated 4 times from hatchling to adult and has been investigated in detail in the free-living nematode, Caenorhabditis elegans. This process is known as moulting and involves numerous enzymes in the synthesis and degradation of the collagenous matrix. The nas-36 and nas-37 genes in C. elegans encode functionally conserved enzymes of the astacin metalloprotease family which, when mutated, result in a phenotype associated with the late-stage moulting defects, namely the inability to remove the preceding cuticle. Extensive genome searches in the gastrointestinal nematode of sheep, Haemonchus contortus, and in the filarial nematode of humans, Brugia malayi, identified NAS-36 but not NAS-37 homologues. Significantly, the nas-36 gene from B. malayi could successfully complement the moult defects associated with C. elegans nas-36, nas-37 and nas-36/nas-37 double mutants, suggesting a conserved function for NAS-36 between these diverse nematode species. This conservation between species was further indicated when the recombinant enzymes demonstrated a similar range of inhibitable metalloprotease activities.

  20. ACUTE FILARIAL INFECTION PRESENTING WITH FITS AND A LTERED SENSORIUM- RARE PRESENTATION. A CASE REPORT

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    Mona

    2013-05-01

    Full Text Available INTRODUCTION: Filarial worms are nematodes that live in lymphatic s and subcutaneous tissues. Eight filarial species are known to infect humans out of which most serious filarial infections are caused mostly by four parasites like Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus and Loa loa. These parasites ar e transmitted by specific species of mosquitoes or other arthropods. The clinical manife stations of filarial diseases develop relatively slowly, these infections should be consi dered to induce chronic diseases with possible long- term debilitating effects. Characteristically , filarial disease is more acute and intense in newly exposed individuals than in natives of endemic areas. [1] Lymphatic filariasis (LF causes lymphoedema, hydrocele and acute attacks of dermato- lymphangio-adenitis. [2] It represents a major public health problem in tropical and subtropical regions of the world. [3] It is mainly a disease of the adult and older age-classes and appear s to be more prevalent in males. [4] Lymphatic filariasis is a major tropical disease aff ecting approximately 120 million people worldwide. India contributes about 40% of the tota l global burden and accounts for about 50% of the people at the risk of infection. A recent sur vey has shown that out of the 25 States/Union territories in India, 22 are endemic and nine state s (Andhra Pradesh, Bihar, Gujarat, Kerala, Maharashtra, Orissa, Tamil Nadu, Utter Pradesh and West Bengal contribute to about 95% of total burden. W. bancrofti is the predominant species accounting for about 98% of the national burden. [5

  1. Vector competence of Aedes aegypti mosquitoes for filarial nematodes is affected by age and nutrient limitation.

    Science.gov (United States)

    Ariani, Cristina V; Juneja, Punita; Smith, Sophia; Tinsley, Matthew C; Jiggins, Francis M

    2015-01-01

    Mosquitoes are one of the most important vectors of human disease. The ability of mosquitoes to transmit disease is dependent on the age structure of the population, as mosquitoes must survive long enough for the parasites to complete their development and infect another human. Age could have additional effects due to mortality rates and vector competence changing as mosquitoes senesce, but these are comparatively poorly understood. We have investigated these factors using the mosquito Aedes aegypti and the filarial nematode Brugia malayi. Rather than observing any effects of immune senescence, we found that older mosquitoes were more resistant, but this only occurred if they had previously been maintained on a nutrient-poor diet of fructose. Constant blood feeding reversed this decline in vector competence, meaning that the number of parasites remained relatively unchanged as mosquitoes aged. Old females that had been maintained on fructose also experienced a sharp spike in mortality after an infected blood meal ("refeeding syndrome") and few survived long enough for the parasite to develop. Again, this effect was prevented by frequent blood meals. Our results indicate that old mosquitoes may be inefficient vectors due to low vector competence and high mortality, but that frequent blood meals can prevent these effects of age.

  2. Absence of Wolbachia endobacteria in Sri Lankan isolates of the nematode parasite of animals Setaria digitata.

    Science.gov (United States)

    Voronin, Denis; Abeykoon, A M L L; Gunawardene, Y I Silva; Dassanayake, Ranil S

    2015-01-30

    Setaria digitata is an animal filarial parasite with natural hosts of cattle and buffaloes that causes mild disease conditions. Infection of non-permissive hosts such as goats, sheep and horses, by this nematode can cause cerebrospinal nematodiasis that leads to lumbar paralysis and the eventual death of the animals and inflicts considerable economic losses on livestock farmers. Wolbachia are obligate mutualistic endosymbionts for some filarial nematodes and are currently being targeted for the control of diseases caused by these parasites. However, little is known about the occurrence of this endosymbiont in the Setariidae family. In this work, worms collected from infected cattle in Sri Lanka were morphologically identified as S. digitata and tested for the presence of Wolbachia by PCR screening using the WSP- and Wolbachia-specific 16S rRNA and multilocus sequence typing primers that were designed to amplify the gatB, coxA, hcpA, ftsZ and fbpA sequences of Wolbachia. The presence of endobacteria in S. digitata was also examined by whole-mount immunofluorescence staining of the parasites and transmission electron microscopic studies. These analyses did not produce evidence of presence of Wolbachia or any other endosymbiotic bacteria in S. digitata, whereas such evidence was found in Brugia malayi, which was used as a positive control in this study.

  3. Minocycline as a re-purposed anti-Wolbachia macrofilaricide: superiority compared with doxycycline regimens in a murine infection model of human lymphatic filariasis.

    Science.gov (United States)

    Sharma, Raman; Al Jayoussi, Ghaith; Tyrer, Hayley E; Gamble, Joanne; Hayward, Laura; Guimaraes, Ana F; Davies, Jill; Waterhouse, David; Cook, Darren A N; Myhill, Laura J; Clare, Rachel H; Cassidy, Andrew; Steven, Andrew; Johnston, Kelly L; Ford, Louise; Turner, Joseph D; Ward, Stephen A; Taylor, Mark J

    2016-03-21

    Lymphatic filariasis and onchocerciasis are parasitic helminth diseases, which cause severe morbidities such as elephantiasis, skin disease and blindness, presenting a major public health burden in endemic communities. The anti-Wolbachia consortium (A·WOL: http://www.a-wol.com/) has identified a number of registered antibiotics that target the endosymbiotic bacterium, Wolbachia, delivering macrofilaricidal activity. Here we use pharmacokinetics/pharmacodynamics (PK/PD) analysis to rationally develop an anti-Wolbachia chemotherapy by linking drug exposure to pharmacological effect. We compare the pharmacokinetics and anti-Wolbachia efficacy in a murine Brugia malayi model of minocycline versus doxycycline. Doxycycline exhibits superior PK in comparison to minocycline resulting in a 3-fold greater exposure in SCID mice. Monte-Carlo simulations confirmed that a bi-daily 25-40 mg/Kg regimen is bioequivalent to a clinically effective 100-200 mg/day dose for these tetracyclines. Pharmacodynamic studies showed that minocycline depletes Wolbachia more effectively than doxycycline (99.51% vs. 90.35%) after 28 day 25 mg/Kg bid regimens with a more potent block in microfilarial production. PK/PD analysis predicts that minocycline would be expected to be 1.7 fold more effective than doxycycline in man despite lower exposure in our infection models. Our findings warrant onward clinical investigations to examine the clinical efficacy of minocycline treatment regimens against lymphatic filariasis and onchocerciasis.

  4. Absence of Wolbachia endobacteria in the non-filariid nematodes Angiostrongylus cantonensis and A. costaricensis

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    Graeff-Teixeira Carlos

    2008-09-01

    Full Text Available Abstract The majority of filarial nematodes harbour Wolbachia endobacteria, including the major pathogenic species in humans, Onchocerca volvulus, Brugia malayi and Wuchereria bancrofti. These obligate endosymbionts have never been demonstrated unequivocally in any non-filariid nematode. However, a recent report described the detection by PCR of Wolbachia in the metastrongylid nematode, Angiostrongylus cantonensis (rat lungworm, a leading cause of eosinophilic meningitis in humans. To address the intriguing possibility of Wolbachia infection in nematode species distinct from the Family Onchocercidae, we used both PCR and immunohistochemistry to screen samples of A. cantonensis and A. costaricensis for the presence of this endosymbiont. We were unable to detect Wolbachia in either species using these methodologies. In addition, bioinformatic and phylogenetic analyses of the Wolbachia gene sequences reported previously from A. cantonensis indicate that they most likely result from contamination with DNA from arthropods and filarial nematodes. This study demonstrates the need for caution in relying solely on PCR for identification of new endosymbiont strains from invertebrate DNA samples.

  5. Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

    Science.gov (United States)

    Guiliano, D; Ganatra, M; Ware, J; Parrot, J; Daub, J; Moran, L; Brennecke, H; Foster, J M; Supali, T; Blaxter, M; Scott, A L; Williams, S A; Slatko, B E

    1999-07-01

    A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.

  6. Helminth genomics: The implications for human health.

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    Paul J Brindley

    Full Text Available More than two billion people (one-third of humanity are infected with parasitic roundworms or flatworms, collectively known as helminth parasites. These infections cause diseases that are responsible for enormous levels of morbidity and mortality, delays in the physical development of children, loss of productivity among the workforce, and maintenance of poverty. Genomes of the major helminth species that affect humans, and many others of agricultural and veterinary significance, are now the subject of intensive genome sequencing and annotation. Draft genome sequences of the filarial worm Brugia malayi and two of the human schistosomes, Schistosoma japonicum and S. mansoni, are now available, among others. These genome data will provide the basis for a comprehensive understanding of the molecular mechanisms involved in helminth nutrition and metabolism, host-dependent development and maturation, immune evasion, and evolution. They are likely also to predict new potential vaccine candidates and drug targets. In this review, we present an overview of these efforts and emphasize the potential impact and importance of these new findings.

  7. VECTORS OF MALARIA AND FILARIASIS IN INDONESIA

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    Hoedojo Hoedojo

    2012-09-01

    Full Text Available Malaria at present is still one of the important mosquito-borne diseases in Indonesia. The disease is widespread all over the country and involves nearly all islands. Sixteen Anopheles species have been reconfirmed as malaria vectors. They were distributed geographi­cally as follows: Coastal areas and lagoons ------------------------------------- An sundaicus and An.subpictus Cultivated ricefields and swampy areas -------------------- An.aconitus, An.barbirostris, An.nigerrimus and An.sinensis Forest inland areas in shaded temporary pools, muddy animal wallows and hoof-prints -------------------------------------------------------- An.balabacensis, An.bancrofti, An.farauti, An.koliensis and An.punctulatus Swamp forest edge in ditches with vegeta- ---------------- An.letifer and An.ludlowae don Hilly areas in seepages, streams and clear moving water ---------------------------------------------- Anflavirostris, An.maculatus and Anminimus.   The species (of most general importance is An.sundaicus, which is restricted by its preference for brackish water and is prevalent in coastal areas of Java. Their types in behaviour of An.sundaicus appear as follows : 1. An.sundaicus in South Coast of Java in general. This species is essentially anthropophilic, exophagic and rests outdoor. It shows susceptible to DDT. 2. An.sundaicus in Cilacap, Central Java. This mosquito is a pure anthropophilic form. It bites man in houses and outdoors, rests indoors and is known resistant to DDT. 3. An.sundaicus in Yogyakarta and Purworejo, Central Java. This mosquito is a strong zoophilic species. It rests and prefers to bite outdoors and shows tolerance to DDT. Human filariasis in Indonesia is the result of infection by three endemic species, namely, Wuchereria bancrofti, Brugia malayi, and Brugia timori.W.bancrofti infection is found in both urban and rural areas. Twenty species of mosquitoes are confirmed as filariasis vectors. The urban type bancroftian filariasis

  8. POTRET VEKTOR MALARIA DAN FILARIASIS DI KECAMATAN SEMBAKUNG KABUPATEN NUNUKAN PROPINSI KALIMANTAN TIMUR

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    Lukman Waris

    2013-12-01

    Full Text Available ABSTRACT The animal resources  infectious diseases, especially malaria and filariasis remains a public health problems, especially in the tropics because the prevalence is quite high. Malaria and filariasis generally invade countries that are developing, including in Indonesia, especially in communities withlow socio-economic in rural areas. Nunukan strategic location in the economic field is relatively favorable for Indonesia, but in the health field area into a region with an epidemiological transition that infectious disease is very high. Some of the potential development into diseases that plague andshowed the higher tendency over the years such as malaria, filariasis, dengue and other infectious diseases. The research was carried out among others to obtain information about the prevalence and the vectors of malaria and filariasis are then used as a material consideration in determining the type and model of malaria prevention and intervention programs filariasis in endemic areas in Nunukan, East Kalimantan, namely District Sembakung. Entomology in malaria research survey in the village of Lubakan Kec. Sembakung in getting An.hyrcanus g and An.barbirostris suspected as vectors of malaria, while the results of parasitological surveys with finger blood preparations obtained positive 97.34% of 188 people suffering frommalaria are examined. Survey research entomologist at the roof of filariasis in the village district. Sembakung in getting suspected mosquito as the vector of filariasis is M.uniformis, M.annulata, M.indiana and Cx.quinquefasciatus while the results of parasitological surveys with blood preparations obtained finger for filariasis, as many as 8 people (3.51% from 288 people who tested positive for Brugia malayi filariasis. The role of various parties are expected to conduct prevention and control of parasitic diseases, not only of health but there is need for cooperation with related agencies such as public employment offices and

  9. Dual RNA-seq of parasite and host reveals gene expression dynamics during filarial worm-mosquito interactions.

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    Young-Jun Choi

    2014-05-01

    Full Text Available BACKGROUND: Parasite biology, by its very nature, cannot be understood without integrating it with that of the host, nor can the host response be adequately explained without considering the activity of the parasite. However, due to experimental limitations, molecular studies of parasite-host systems have been predominantly one-sided investigations focusing on either of the partners involved. Here, we conducted a dual RNA-seq time course analysis of filarial worm parasite and host mosquito to better understand the parasite processes underlying development in and interaction with the host tissue, from the establishment of infection to the development of infective-stage larva. METHODOLOGY/PRINCIPAL FINDINGS: Using the Brugia malayi-Aedes aegypti system, we report parasite gene transcription dynamics, which exhibited a highly ordered developmental program consisting of a series of cyclical and state-transitioning temporal patterns. In addition, we contextualized these parasite data in relation to the concurrent dynamics of the host transcriptome. Comparative analyses using uninfected tissues and different host strains revealed the influence of parasite development on host gene transcription as well as the influence of the host environment on parasite gene transcription. We also critically evaluated the life-cycle transcriptome of B. malayi by comparing developmental stages in the mosquito relative to those in the mammalian host, providing insight into gene expression changes underpinning the mosquito-borne parasitic lifestyle of this heteroxenous parasite. CONCLUSIONS/SIGNIFICANCE: The data presented herein provide the research community with information to design wet lab experiments and select candidates for future study to more fully dissect the whole set of molecular interactions of both organisms in this mosquito-filarial worm symbiotic relationship. Furthermore, characterization of the transcriptional program over the complete life cycle of

  10. Detection of circulating parasite-derived microRNAs in filarial infections.

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    Lucienne Tritten

    2014-07-01

    Full Text Available Filarial nematodes cause chronic and profoundly debilitating diseases in both humans and animals. Applications of novel technology are providing unprecedented opportunities to improve diagnosis and our understanding of the molecular basis for host-parasite interactions. As a first step, we investigated the presence of circulating miRNAs released by filarial nematodes into the host bloodstream. miRNA deep-sequencing combined with bioinformatics revealed over 200 mature miRNA sequences of potential nematode origin in Dirofilaria immitis-infected dog plasma in two independent analyses, and 21 in Onchocerca volvulus-infected human serum. Total RNA obtained from D. immitis-infected dog plasma was subjected to stem-loop RT-qPCR assays targeting two detected miRNA candidates, miR-71 and miR-34. Additionally, Brugia pahangi-infected dog samples were included in the analysis, as these miRNAs were previously detected in extracts prepared from this species. The presence of miR-71 and miR-34 discriminated infected samples (both species from uninfected samples, in which no specific miRNA amplification occurred. However, absolute miRNA copy numbers were not significantly correlated with microfilaraemia for either parasite. This may be due to the imprecision of mf counts to estimate infection intensity or to miRNA contributions from the unknown number of adult worms present. Nonetheless, parasite-derived circulating miRNAs are found in plasma or serum even for those species that do not live in the bloodstream.

  11. Detection of circulating parasite-derived microRNAs in filarial infections.

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    Tritten, Lucienne; Burkman, Erica; Moorhead, Andrew; Satti, Mohammed; Geary, James; Mackenzie, Charles; Geary, Timothy

    2014-07-01

    Filarial nematodes cause chronic and profoundly debilitating diseases in both humans and animals. Applications of novel technology are providing unprecedented opportunities to improve diagnosis and our understanding of the molecular basis for host-parasite interactions. As a first step, we investigated the presence of circulating miRNAs released by filarial nematodes into the host bloodstream. miRNA deep-sequencing combined with bioinformatics revealed over 200 mature miRNA sequences of potential nematode origin in Dirofilaria immitis-infected dog plasma in two independent analyses, and 21 in Onchocerca volvulus-infected human serum. Total RNA obtained from D. immitis-infected dog plasma was subjected to stem-loop RT-qPCR assays targeting two detected miRNA candidates, miR-71 and miR-34. Additionally, Brugia pahangi-infected dog samples were included in the analysis, as these miRNAs were previously detected in extracts prepared from this species. The presence of miR-71 and miR-34 discriminated infected samples (both species) from uninfected samples, in which no specific miRNA amplification occurred. However, absolute miRNA copy numbers were not significantly correlated with microfilaraemia for either parasite. This may be due to the imprecision of mf counts to estimate infection intensity or to miRNA contributions from the unknown number of adult worms present. Nonetheless, parasite-derived circulating miRNAs are found in plasma or serum even for those species that do not live in the bloodstream.

  12. Diarylheptanoid compounds from Alnus nepalensis express in vitro and in vivo antifilarial activity.

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    Yadav, Deepti; Kushwaha, Vikas; Saxena, Kirti; Verma, Richa; Murthy, P Kalpana; Gupta, Madan M

    2013-12-01

    A large number of medicinal plants remain to be explored for antifilarial compounds. In the present study a crude methanolic extract of leaves of Alnus nepalensis, chloroform- and n-butanol-partitioned fractions from the crude extract and 6 bioactivity-guided isolated compounds including two new diarylheptanoid from the fractions were assayed for microfilaricidal, macrofilaricidal and female worm sterilizing activity using the lymphatic filariid Brugia malayi in in vitro and in vivo systems. In vitro, the crude methanolic extract exerted better microfilaricidal (LC100: 15.63μg/ml, IC50: 6.00μg/ml) than macrofilaricidal (LC100: >250; IC50: 88μg/ml) activity whereas chloroform and n-butanol fractions were more macrofilaricidal (LC100: 125 and 31.25μg/ml; IC50: 13.14 and 11.84, respectively) than microfilaricidal (LC100: 250-500μg/ml, IC50: 44.16μg/ml). In addition, n-butanol fraction also caused 74% inhibition in MTT reduction potential of the adult worms. In vivo (doses: crude: 100-200mg/kg; fractions: 100mg/kg, i.p.×5 days) the chloroform fraction exerted >50% macrofilaricidal activity whereas methanolic extract and n-butanol fraction produced 38-40% macrofilaricidal action along with some female sterilizing efficacy. Of the 5 diarylheptanoid compounds isolated, alnus dimer, and (5S)-5-hydroxy-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-3-heptanone were found to show the most potent with both macrofilaricidal (LC100: 15.63μg/ml, IC50: 6.57-10.31μg/ml) and microfilaricidal (LC100: 31.25-62.5μg/ml, IC50: 11.05-22.10μg/ml) activity in vitro. These findings indicate that the active diarylheptanoid compounds may provide valuable lead for design and development of new antifilarial agent(s).

  13. Heterologous expression of the filarial nematode alt gene products reveals their potential to inhibit immune function

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    Aebischer Toni

    2005-03-01

    Full Text Available Abstract Background Parasites exploit sophisticated strategies to evade host immunity that require both adaptation of existing genes and evolution of new gene families. We have addressed this question by testing the immunological function of novel genes from helminth parasites, in which conventional transgenesis is not yet possible. We investigated two such novel genes from Brugia malayi termed abundant larval transcript (alt, expression of which reaches ~5% of total transcript at the time parasites enter the human host. Results To test the hypothesis that ALT proteins modulate host immunity, we adopted an alternative transfection strategy to express these products in the protozoan parasite Leishmania mexicana. We then followed the course of infection in vitro in macrophages and in vivo in mice. Expression of ALT proteins, but not a truncated mutant, conferred greater infectivity of macrophages in vitro, reaching 3-fold higher parasite densities. alt-transfected parasites also caused accelerated disease in vivo, and fewer mice were able to clear infection of organisms expressing ALT. alt-transfected parasites were more resistant to IFN-γ-induced killing by macrophages. Expression profiling of macrophages infected with transgenic L. mexicana revealed consistently higher levels of GATA-3 and SOCS-1 transcripts, both associated with the Th2-type response observed in in vivo filarial infection. Conclusion Leishmania transfection is a tractable and informative approach to determining immunological functions of single genes from heterologous organisms. In the case of the filarial ALT proteins, our data suggest that they may participate in the Th2 bias observed in the response to parasite infection by modulating cytokine-induced signalling within immune system cells.

  14. Search for new prototypes for the chemotherapy of filariasis: a chemotherapeutic and biochemical approach.

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    Bajpai, Preeti; Verma, Shailendra Kumar; Katiyar, Diksha; Tewari, Neetu; Tripathi, R P; Bansal, Iti; Saxena, J K; Misra-Bhattacharya, Shailja

    2005-04-01

    The antifilarial activity of two coumarin derivatives (A, B) and three glycosyl amine derivatives (D, E, F) was evaluated against a subperiodic strain of human lymphatic filarial parasite Brugia malayi by the intraperitoneal route at 50 mg/kg for 5 consecutive days. Of these, the two sugar derivatives (D and E) were selected for evaluation by the oral route based on their microfilaricidal (mild), macrofilaricidal and female worm sterilization efficacy using the i.p. route of administration. Compound E was finally selected for combination therapy on the basis of its microfilaricidal and embryostatic action by the oral route and its spectrum of activity against micro- and macrofilariae including embryostatic activity by the i.p. route. In addition, E also significantly inhibited the parasite DNA topoisomerase II. Compound A, in contrast, led to an enhanced adult worm burden. Compound B was toxic by the i.p. route, killing all of the treated animals before completion of the experiment. Some of these compounds demonstrated significant antifilarial efficacy of varying degree when tested in vitro Compounds B, D and F also killed adult B. malayi in vitro at 100 muM while 50 muM resulted in very slow motility of worms. Compound E in combination with a promising macrofilaricidal benzopyran derivative reported by us recently (compound C) did not show any synergistic or additive effect. These two compounds (C and E) individually on oral administration with either DEC or ivermectin significantly improved microfilaricidal efficacy in terms of intensity and duration of suppressed microfilaraemia. The combination of DEC with compound E demonstrated marginal enhancement in adulticidal efficacy, however, the embryostatic effect of the duo was significantly higher than that exerted by the individual agents. It may thus be inferred that in the absence of an adulticidal antifilarial drug, the use of potential antifilarials in combination with the standard filaricides may yield better

  15. In vitro, in silico and in vivo studies of ursolic acid as an anti-filarial agent.

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    Kalani, Komal; Kushwaha, Vikas; Sharma, Pooja; Verma, Richa; Srivastava, Mukesh; Khan, Feroz; Murthy, P K; Srivastava, Santosh Kumar

    2014-01-01

    As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC100: 50; IC50: 8.84 µM) and female adult worms (LC100: 100; IC50: 35.36 µM) as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (-8.6) with respect to the standard antifilarial drugs, ivermectin (IVM -8.4) and diethylcarbamazine (DEC-C -4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection), which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuter's 'Metadrug' tool screening predictions.

  16. In vitro, in silico and in vivo studies of ursolic acid as an anti-filarial agent.

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    Komal Kalani

    Full Text Available As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC100: 50; IC50: 8.84 µM and female adult worms (LC100: 100; IC50: 35.36 µM as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock scores for UA (-8.6 with respect to the standard antifilarial drugs, ivermectin (IVM -8.4 and diethylcarbamazine (DEC-C -4.6 on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection, which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuter's 'Metadrug' tool screening predictions.

  17. Identification of attractive drug targets in neglected-disease pathogens using an in silico approach.

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    Gregory J Crowther

    Full Text Available BACKGROUND: The increased sequencing of pathogen genomes and the subsequent availability of genome-scale functional datasets are expected to guide the experimental work necessary for target-based drug discovery. However, a major bottleneck in this has been the difficulty of capturing and integrating relevant information in an easily accessible format for identifying and prioritizing potential targets. The open-access resource TDRtargets.org facilitates drug target prioritization for major tropical disease pathogens such as the mycobacteria Mycobacterium leprae and Mycobacterium tuberculosis; the kinetoplastid protozoans Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi; the apicomplexan protozoans Plasmodium falciparum, Plasmodium vivax, and Toxoplasma gondii; and the helminths Brugia malayi and Schistosoma mansoni. METHODOLOGY/PRINCIPAL FINDINGS: Here we present strategies to prioritize pathogen proteins based on whether their properties meet criteria considered desirable in a drug target. These criteria are based upon both sequence-derived information (e.g., molecular mass and functional data on expression, essentiality, phenotypes, metabolic pathways, assayability, and druggability. This approach also highlights the fact that data for many relevant criteria are lacking in less-studied pathogens (e.g., helminths, and we demonstrate how this can be partially overcome by mapping data from homologous genes in well-studied organisms. We also show how individual users can easily upload external datasets and integrate them with existing data in TDRtargets.org to generate highly customized ranked lists of potential targets. CONCLUSIONS/SIGNIFICANCE: Using the datasets and the tools available in TDRtargets.org, we have generated illustrative lists of potential drug targets in seven tropical disease pathogens. While these lists are broadly consistent with the research community's current interest in certain specific proteins, and suggest

  18. Analysis of nematode motion using an improved light-scatter based system.

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    Chuck S Nutting

    2015-02-01

    Full Text Available The detailed assessment of nematode activity and viability still remains a relatively undeveloped area of biological and medical research. Computer-based approaches to assessing the motility of larger nematode stages have been developed, yet these lack the capability to detect and analyze the more subtle and important characteristics of the motion of nematodes. There is currently a need to improved methods of assessing the viability and health of parasitic worms.We describe here a system that converts the motion of nematodes through a light-scattering system into an electrical waveform, and allows for reproducible, and wholly non-subjective, assessment of alterations in motion, as well as estimation of the number of nematode worms of different forms and sizes. Here we have used Brugia sp. microfilariae (L1, infective larvae (L3 and adults, together with the free-living nematode Caenorhabditis elegans.The motion of worms in a small (200 ul volume can be detected, with the presence of immotile worms not interfering with the readings at practical levels (up to at least 500 L1 /200 ul. Alterations in the frequency of parasite movement following the application of the anti-parasitic drugs, (chloroquine and imatinib; the anti-filarial effect of the latter agent is the first demonstrated here for the first time. This system can also be used to estimate the number of parasites, and shortens the time required to estimate parasites numbers, and eliminates the need for microscopes and trained technicians to provide an estimate of microfilarial sample sizes up to 1000 parasites/ml. Alterations in the form of motion of the worms can also be depicted.This new instrument, named a "WiggleTron", offers exciting opportunities to further study nematode biology and to aid drug discovery, as well as contributing to a rapid estimate of parasite numbers in various biological samples.

  19. High pressure freezing/freeze substitution fixation improves the ultrastructural assessment of Wolbachia endosymbiont-filarial nematode host interaction.

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    Kerstin Fischer

    Full Text Available BACKGROUND: Wolbachia α-proteobacteria are essential for growth, reproduction and survival for many filarial nematode parasites of medical and veterinary importance. Endobacteria were discovered in filarial parasites by transmission electron microscopy in the 1970's using chemically fixed specimens. Despite improvements of fixation and electron microscopy techniques during the last decades, methods to study the Wolbachia/filaria interaction on the ultrastructural level remained unchanged and the mechanisms for exchange of materials and for motility of endobacteria are not known. METHODOLOGY/PRINCIPAL FINDING: We used high pressure freezing/freeze substitution to improve fixation of Brugia malayi and its endosymbiont, and this led to improved visualization of different morphological forms of Wolbachia. The three concentric, bilayer membranes that surround the endobacterial cytoplasm were well preserved. Vesicles with identical membrane structures were identified close to the endobacteria, and multiple bacteria were sometimes enclosed within a single outer membrane. Immunogold electron microscopy using a monoclonal antibody directed against Wolbachia surface protein-1 labeled the membranes that enclose Wolbachia and Wolbachia-associated vesicles. High densities of Wolbachia were observed in the lateral chords of L4 larvae, immature, and mature adult worms. Extracellular Wolbachia were sometimes present in the pseudocoelomic cavity near the developing female reproductive organs. Wolbachia-associated actin tails were not observed. Wolbachia motility may be explained by their residence within vacuoles, as they may co-opt the host cell's secretory pathway to move within and between cells. CONCLUSIONS/SIGNIFICANCE: High pressure freezing/freeze substitution significantly improved the preservation of filarial tissues for electron microscopy to reveal membranes and sub cellular structures that could be crucial for exchange of materials between Wolbachia

  20. Impact of six rounds of mass drug administration on Brugian filariasis and soil-transmitted helminth infections in eastern Indonesia.

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    Taniawati Supali

    Full Text Available BACKGROUND: The lymphatic filarial parasite Brugia timori occurs only in eastern Indonesia where it causes high morbidity. The absence of an animal reservoir, the inefficient transmission by Anopheles mosquitoes and the high sensitivity to DEC/albendazole treatment make this species a prime candidate for elimination by mass drug administration (MDA. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the effect of MDA using DEC and albendazole on B. timori and soil transmitted helminths (STH in a cross-sectional study of a sentinel village on Alor Island annually over a period of 10 years. Pre-MDA the microfilaria (MF prevalence was 26% and 80% of the residents had filaria-specific IgG4 antibodies. In 2010, 34 months after the 6(th round of MDA, MF and antibody rates were only 0.17% and 6.4%, respectively. The MDA campaign had also a beneficial effect on STH. Baseline prevalence rates for Ascaris, hookworm and Trichuris were 34%, 28%, and 11%, respectively; these rates were reduced to 27%, 4%, and 2% one year after the 5(th round of MDA. Unfortunately, STH rates rebounded 34 months after cessation of MDA and approached pre-MDA rates. However, the intensity of STH infection in 2009 was still reduced, and no heavy infections were detected. CONCLUSIONS/SIGNIFICANCE: MDA with DEC/albendazole has had a major impact on B. timori MF and IgG4 antibody rates, providing a proof of principle that elimination is feasible. We also documented the value of annual DEC/albendazole as a mass de-worming intervention and the importance of continuing some form of STH control after cessation of MDA for filariasis.

  1. A survey of canine filarial diseases of veterinary and public health significance in India

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    McInnes Linda M

    2010-04-01

    Full Text Available Abstract Background Dirofilaria spp., Acanthocheilonema spp. and Brugia spp. have all been reported in Indian dogs. In previous studies, diagnosis was made by morphological identification only. This is the first geographically stratified cross-sectional study in India to determine the prevalence and geographical distribution of canine filarial species of veterinary and public health importance, using a combination of conventional and molecular diagnostic techniques. Results A total of 139 from 525 dogs (26.5%; 95% CI 22.7, 30.3 were positive for microfilariae. The most common species of canine filaria identified in this study was A. reconditum (9.3% followed by D. repens (6.7% and D. immitis (1.5%. Three out of 525 dogs were found to have mixed infections on PCR. The morphological and molecular evidence on the sequence of the 18S gene and phylogenetic analysis of the ITS-2 region provided strong evidence that the canine microfilariae discovered in the Himalayan city of Ladakh belong to a novel species of Acanthocheilonema. Two dogs in Ladakh were also found to have mixed infections of the novel species described above and a unique microfilaria which morphologically resembled Microfilaria auquieri Foley, 1921. Conclusions At least six species of filarial nematode are now known to infect dogs in India, two of which were reported for the first time in this study. The study also confirms and extends the geographical distribution of canine heartworm (D. immitis which overlaps with D. repens, emphasising the importance for veterinary clinicians and diagnostic laboratories to utilise immunodiagnostic tests that will not cross-react between those two filarial species. From a public health viewpoint, the distribution and prevalences of these nematodes warrant an appropriate prophylaxis to be administered to dogs.

  2. Transcriptomic and proteomic analyses of a Wolbachia-free filarial parasite provide evidence of trans-kingdom horizontal gene transfer.

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    Samantha N McNulty

    Full Text Available Most filarial parasites in the subfamilies Onchocercinae and Dirofilariinae depend on Wolbachia endobacteria to successfully carry out their life cycle. Recently published data indicate that the few Wolbachia-free species in these subfamilies were infected in the distant past and have subsequently shed their endosymbionts. We used an integrated transcriptomic and proteomic analysis of Onchocerca flexuosa to explore the molecular mechanisms that allow worms of this species to survive without a bacterial partner. Roche/454 sequencing of the adult transcriptome produced 16,814 isogroup and 47,252 singleton sequences that are estimated to represent approximately 41% of the complete gene set. Sequences similar to 97 Wolbachia genes were identified from the transcriptome, some of which appear on the same transcripts as sequences similar to nematode genes. Computationally predicted peptides, including those with similarity to Wolbachia proteins, were classified at the domain and pathway levels in order to assess the metabolic capabilities of O. flexuosa and compare against the Wolbachia-dependent model filaria, Brugia malayi. Transcript data further facilitated a shotgun proteomic analysis of O. flexuosa adult worm lysate, resulting in the identification of 1,803 proteins. Three of the peptides detected by mass spectroscopy map to two ABC transport-related proteins from Wolbachia. Antibodies raised to one of the Wolbachia-like peptides labeled a single 38 kDa band on Western blots of O. flexuosa lysate and stained specific worm tissues by immunohistology. Future studies will be required to determine the exact functions of Wolbachia-like peptides and proteins in O. flexuosa and to assess their roles in worm biology.

  3. New features on the moults and morphogenesis of the human filaria Loa loa by using rodent hosts consequences.

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    Bain, O; Wanji, S; Enyong, P; Petit, G; Noireau, F; Eberhard, M I; Wahl, G

    1998-03-01

    The development of the human filaria Loa loa (Dirofilariinae, Onchocercidae), previously studied in monkeys, was studied using the non permissive hosts-mice and jirds. The development proved to be rapid: moult 3 occurred on day 8 post-inoculation, the adult stage was reached on day 25 and measured at that time 3-3.5 mm in length. As in the other filarioids, the female genital apparatus developed during the fourth stage. A critical analysis of the studies on the development of Onchocercid species was made. The optimal duration of the stages (i.g. the shortest time) was chosen for the comparison. It appeared that the duration of the stage 3 was a constant character in a given species whatever the experimental conditions, whereas moult 4 might be retarded in a non susceptible host. Comparison between the 18 developmental cycles of Onchocercidae in the vertebrate host was made. Two biological types could be distinguished: either the moult 3 occurred on day 2-3 and was followed apparently by a late moult 4 (> or = 50 days), or the moult 3 occurred after about one week of development and it was associated with a less long stage 4 (20-40 days). The first group includes Dirofilaria and Onchocerca, the second group brings together mainly Loa and the Onchocercinae of the Dipetalonema line and related genera (Acanthocheilonema, Brugia, Litomosoides, etc.). The groups thus formed suggest real relationships as they fit with the morphology of the infective stage and the results of a recent molecular analysis of the 5S DNA.

  4. Reciprocal immunomodulatory effects of gamma interferon and interleukin-4 on filaria-induced airway hyperresponsiveness.

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    Mehlotra, R K; Hall, L R; Haxhiu, M A; Pearlman, E

    2001-03-01

    Tropical pulmonary eosinophilia (TPE) is a severe asthmatic syndrome of lymphatic filariasis, in which an allergic response is induced to microfilariae (Mf) in the lungs. Previously, in a murine model for TPE, we have demonstrated that recombinant interleukin-12 (IL-12) suppresses pulmonary eosinophilia and airway hyperresponsiveness (AHR) by modulating the T helper (Th) response in the lungs from Th2- to Th1-like, with elevated gamma-interferon (IFN-gamma) production and decreased IL-4 and IL-5 production. The present study examined the immunomodulatory roles of IL-4 and IFN-gamma in filaria-induced AHR and pulmonary inflammation using mice genetically deficient in these cytokines. C57BL/6, IL-4 gene knockout (IL-4(-/-)), and IFN-gamma(-/-) mice were first immunized with soluble Brugia malayi antigens and then inoculated intravenously with 200,000 live Mf. Compared with C57BL/6 mice, IL-4(-/-) mice exhibited significantly reduced AHR, whereas IFN-gamma(-/-) mice had increased AHR. Histopathologically, each mouse strain showed increased cellular infiltration into the lung parenchyma and bronchoalveolar space compared with naïve animals. However, consistent with changes in AHR, IL-4(-/-) mice had less inflammation than C57BL/6 mice, whereas IFN-gamma(-/-) mice had exacerbated pulmonary inflammation with the loss of pulmonary architecture. Systemically, IL-4(-/-) mice produced significantly higher IFN-gamma levels compared with C57BL/6 mice, whereas IFN-gamma(-/-) mice produced significantly higher IL-4 levels. These data indicate that IL-4 is required for the induction of filaria-induced AHR, whereas IFN-gamma suppresses AHR.

  5. Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential.

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    Stéphane Bellafiore

    2008-10-01

    Full Text Available The root knot nematode, Meloidogyne incognita, is an obligate parasite that causes significant damage to a broad range of host plants. Infection is associated with secretion of proteins surrounded by proliferating cells. Many parasites are known to secrete effectors that interfere with plant innate immunity, enabling infection to occur; they can also release pathogen-associated molecular patterns (PAMPs, e.g., flagellin that trigger basal immunity through the nematode stylet into the plant cell. This leads to suppression of innate immunity and reprogramming of plant cells to form a feeding structure containing multinucleate giant cells. Effectors have generally been discovered using genetics or bioinformatics, but M. incognita is non-sexual and its genome sequence has not yet been reported. To partially overcome these limitations, we have used mass spectrometry to directly identify 486 proteins secreted by M. incognita. These proteins contain at least segmental sequence identity to those found in our 3 reference databases (published nematode proteins; unpublished M. incognita ESTs; published plant proteins. Several secreted proteins are homologous to plant proteins, which they may mimic, and they contain domains that suggest known effector functions (e.g., regulating the plant cell cycle or growth. Others have regulatory domains that could reprogram cells. Using in situ hybridization we observed that most secreted proteins were produced by the subventral glands, but we found that phasmids also secreted proteins. We annotated the functions of the secreted proteins and classified them according to roles they may play in the development of root knot disease. Our results show that parasite secretomes can be partially characterized without cognate genomic DNA sequence. We observed that the M. incognita secretome overlaps the reported secretome of mammalian parasitic nematodes (e.g., Brugia malayi, suggesting a common parasitic behavior and a possible

  6. Eosinophils are important for protection, immunoregulation and pathology during infection with nematode microfilariae.

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    Emma T Cadman

    2014-03-01

    Full Text Available Eosinophil responses typify both allergic and parasitic helminth disease. In helminthic disease, the role of eosinophils can be both protective in immune responses and destructive in pathological responses. To investigate whether eosinophils are involved in both protection and pathology during filarial nematode infection, we explored the role of eosinophils and their granule proteins, eosinophil peroxidase (EPO and major basic protein-1 (MBP-1, during infection with Brugia malayi microfilariae. Using eosinophil-deficient mice (PHIL, we further clarify the role of eosinophils in clearance of microfilariae during primary, but not challenge infection in vivo. Deletion of EPO or MBP-1 alone was insufficient to abrogate parasite clearance suggesting that either these molecules are redundant or eosinophils act indirectly in parasite clearance via augmentation of other protective responses. Absence of eosinophils increased mast cell recruitment, but not other cell types, into the broncho-alveolar lavage fluid during challenge infection. In addition absence of eosinophils or EPO alone, augmented parasite-induced IgE responses, as measured by ELISA, demonstrating that eosinophils are involved in regulation of IgE. Whole body plethysmography indicated that nematode-induced changes in airway physiology were reduced in challenge infection in the absence of eosinophils and also during primary infection in the absence of EPO alone. However lack of eosinophils or MBP-1 actually increased goblet cell mucus production. We did not find any major differences in cytokine responses in the absence of eosinophils, EPO or MBP-1. These results reveal that eosinophils actively participate in regulation of IgE and goblet cell mucus production via granule secretion during nematode-induced pathology and highlight their importance both as effector cells, as damage-inducing cells and as supervisory cells that shape both innate and adaptive immunity.

  7. Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification.

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    Catherine B Poole

    Full Text Available Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4, was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg in 25-30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal.

  8. A target repurposing approach identifies N-myristoyltransferase as a new candidate drug target in filarial nematodes.

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    Brendan D Galvin

    2014-09-01

    Full Text Available Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT, an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC50 values of 2.5-10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis.

  9. Cofactor-independent phosphoglycerate mutase from nematodes has limited druggability, as revealed by two high-throughput screens.

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    Crowther, Gregory J; Booker, Michael L; He, Min; Li, Ting; Raverdy, Sylvine; Novelli, Jacopo F; He, Panqing; Dale, Natalie R G; Fife, Amy M; Barker, Robert H; Kramer, Martin L; Van Voorhis, Wesley C; Carlow, Clotilde K S; Wang, Ming-Wei

    2014-01-01

    Cofactor-independent phosphoglycerate mutase (iPGAM) is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in parasitic nematodes such as Brugia malayi, a cause of lymphatic filariasis (LF). iPGAM's active site is small and hydrophilic, implying that it may not be druggable, but another binding site might permit allosteric inhibition. As a comprehensive assessment of iPGAM's druggability, high-throughput screening (HTS) was conducted at two different locations: ∼220,000 compounds were tested against the C. elegans iPGAM by Genzyme Corporation, and ∼160,000 compounds were screened against the B. malayi iPGAM at the National Center for Drug Screening in Shanghai. iPGAM's catalytic activity was coupled to downstream glycolytic enzymes, resulting in NADH consumption, as monitored by a decline in visible-light absorbance at 340 nm. This assay performed well in both screens (Z'-factor >0.50) and identified two novel inhibitors that may be useful as chemical probes. However, these compounds have very modest potency against the B. malayi iPGAM (IC50 >10 µM) and represent isolated singleton hits rather than members of a common scaffold. Thus, despite the other appealing properties of the nematode iPGAMs, their low druggability makes them challenging to pursue as drug targets. This study illustrates a "druggability paradox" of target-based drug discovery: proteins are generally unsuitable for resource-intensive HTS unless they are considered druggable, yet druggability is often difficult to predict in the absence of HTS data.

  10. A target repurposing approach identifies N-myristoyltransferase as a new candidate drug target in filarial nematodes.

    Science.gov (United States)

    Galvin, Brendan D; Li, Zhiru; Villemaine, Estelle; Poole, Catherine B; Chapman, Melissa S; Pollastri, Michael P; Wyatt, Paul G; Carlow, Clotilde K S

    2014-09-01

    Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC50 values of 2.5-10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis.

  11. Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Poole, Catherine B; Ettwiller, Laurence; Tanner, Nathan A; Evans, Thomas C; Wanji, Samuel; Carlow, Clotilde K S

    2015-01-01

    Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25-30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal.

  12. Cofactor-independent phosphoglycerate mutase from nematodes has limited druggability, as revealed by two high-throughput screens.

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    Gregory J Crowther

    Full Text Available Cofactor-independent phosphoglycerate mutase (iPGAM is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in parasitic nematodes such as Brugia malayi, a cause of lymphatic filariasis (LF. iPGAM's active site is small and hydrophilic, implying that it may not be druggable, but another binding site might permit allosteric inhibition. As a comprehensive assessment of iPGAM's druggability, high-throughput screening (HTS was conducted at two different locations: ∼220,000 compounds were tested against the C. elegans iPGAM by Genzyme Corporation, and ∼160,000 compounds were screened against the B. malayi iPGAM at the National Center for Drug Screening in Shanghai. iPGAM's catalytic activity was coupled to downstream glycolytic enzymes, resulting in NADH consumption, as monitored by a decline in visible-light absorbance at 340 nm. This assay performed well in both screens (Z'-factor >0.50 and identified two novel inhibitors that may be useful as chemical probes. However, these compounds have very modest potency against the B. malayi iPGAM (IC50 >10 µM and represent isolated singleton hits rather than members of a common scaffold. Thus, despite the other appealing properties of the nematode iPGAMs, their low druggability makes them challenging to pursue as drug targets. This study illustrates a "druggability paradox" of target-based drug discovery: proteins are generally unsuitable for resource-intensive HTS unless they are considered druggable, yet druggability is often difficult to predict in the absence of HTS data.

  13. Lipoprotein biosynthesis as a target for anti-Wolbachia treatment of filarial nematodes

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    Slatko Barton E

    2010-10-01

    Full Text Available Abstract Background Lymphatic filariasis and onchocerciasis are debilitating diseases caused by filarial nematodes. Disease pathogenesis is induced by inflammatory responses following the death of the parasite. Wolbachia endosymbionts of filariae are potent inducers of innate and adaptive inflammation and bacterial lipoproteins have been identified as the ligands that bind toll-like receptors (TLR 2 and TLR6. Lipoproteins are important structural and functional components of bacteria and therefore enzymes involved in Wolbachia lipoprotein biosynthesis are potential chemotherapeutic targets. Results Globomycin, a signal peptidase II (LspA inhibitor, has activity against Gram-negative bacteria and a putative lspA gene has been identified from the Wolbachia genome of Brugia malayi (wBm. The amino acids required for function are strictly conserved and functionality was verified by complementation tests in a temperature-sensitive Escherichia coli lspA mutant. Also, transformation of wild type E. coli with Wolbachia lspA conferred significant globomycin resistance. A cell-based screen has been developed utilizing a Wolbachia-containing Aedes albopictus cell line to assay novel compounds active against Wolbachia. Globomycin was screened using this assay, which resulted in a dose-dependent reduction in Wolbachia load. Furthermore, globomycin was also effective in reducing the motility and viability of adult B. malayi in vitro. Conclusions These studies validate lipoprotein biosynthesis as a target in an organism for which no genetic tools are available. Further studies to evaluate drugs targeting this pathway are underway as part of the A-WOL drug discovery and development program.

  14. Utilization of computer processed high definition video imaging for measuring motility of microscopic nematode stages on a quantitative scale: “The Worminator”

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    Bob Storey

    2014-12-01

    Full Text Available A major hindrance to evaluating nematode populations for anthelmintic resistance, as well as for screening existing drugs, new compounds, or bioactive plant extracts for anthelmintic properties, is the lack of an efficient, objective, and reproducible in vitro assay that is adaptable to multiple life stages and parasite genera. To address this need we have developed the “Worminator” system, which objectively and quantitatively measures the motility of microscopic stages of parasitic nematodes. The system is built around the computer application “WormAssay”, developed at the Center for Discovery and Innovation in Parasitic Diseases at the University of California, San Francisco. WormAssay was designed to assess motility of macroscopic parasites for the purpose of high throughput screening of potential anthelmintic compounds, utilizing high definition video as an input to assess motion of adult stage (macroscopic parasites (e.g. Brugia malayi. We adapted this assay for use with microscopic parasites by modifying the software to support a full frame analysis mode that applies the motion algorithm to the entire video frame. Thus, the motility of all parasites in a given well are recorded and measured simultaneously. Assays performed on third-stage larvae (L3 of the bovine intestinal nematode Cooperia spp., as well as microfilariae (mf of the filarioid nematodes B. malayi and Dirofilaria immitis, yielded reproducible dose responses using the macrocyclic lactones ivermectin, doramectin, and moxidectin, as well as the nicotinic agonists, pyrantel, oxantel, morantel, and tribendimidine. This new computer based-assay is simple to use, requires minimal new investment in equipment, is robust across nematode genera and developmental stage, and does not require subjective scoring of motility by an observer. Thus, the “Worminator” provides a relatively low-cost platform for developing genera- and stage-specific assays with high efficiency and

  15. Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™

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    Luck, Ashley N.; Slatko, Barton E.; Foster, Jeremy M.

    2017-01-01

    Efficient transcriptomic sequencing of microbial mRNA derived from host-microbe associations is often compromised by the much lower relative abundance of microbial RNA in the mixed total RNA sample. One solution to this problem is to perform extensive sequencing until an acceptable level of transcriptome coverage is obtained. More cost-effective methods include use of prokaryotic and/or eukaryotic rRNA depletion strategies, sometimes in conjunction with depletion of polyadenylated eukaryotic mRNA. Here, we report use of Cappable-seq™ to specifically enrich, in a single step, Wolbachia endobacterial mRNA transcripts from total RNA prepared from the parasitic filarial nematode, Brugia malayi. The obligate Wolbachia endosymbiont is a proven drug target for many human filarial infections, yet the precise nature of its symbiosis with the nematode host is poorly understood. Insightful analysis of the expression levels of Wolbachia genes predicted to underpin the mutualistic association and of known drug target genes at different life cycle stages or in response to drug treatments is typically challenged by low transcriptomic coverage. Cappable-seq resulted in up to ~ 5-fold increase in the number of reads mapping to Wolbachia. On average, coverage of Wolbachia transcripts from B. malayi microfilariae was enriched ~40-fold by Cappable-seq. Additionally, this method has an additional benefit of selectively removing abundant prokaryotic ribosomal RNAs.The deeper microbial transcriptome sequencing afforded by Cappable-seq facilitates more detailed characterization of gene expression levels of pathogens and symbionts present in animal tissues. PMID:28291780

  16. Pan-phylum Comparison of Nematode Metabolic Potential.

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    Rahul Tyagi

    2015-05-01

    Full Text Available Nematodes are among the most important causative pathogens of neglected tropical diseases. The increased availability of genomic and transcriptomic data for many understudied nematode species provides a great opportunity to investigate different aspects of their biology. Increasingly, metabolic potential of pathogens is recognized as a critical determinant governing their development, growth and pathogenicity. Comparing metabolic potential among species with distinct trophic ecologies can provide insights on overall biology or molecular adaptations. Furthermore, ascertaining gene expression at pathway level can help in understanding metabolic dynamics over development. Comparison of biochemical pathways (or subpathways, i.e. pathway modules among related species can also retrospectively indicate potential mistakes in gene-calling and functional annotation. We show with numerous illustrative case studies that comparisons at the level of pathway modules have the potential to uncover biological insights while remaining computationally tractable. Here, we reconstruct and compare metabolic modules found in the deduced proteomes of 13 nematodes and 10 non-nematode species (including hosts of the parasitic nematode species. We observed that the metabolic potential is, in general, concomitant with phylogenetic and/or ecological similarity. Varied metabolic strategies are required among the nematodes, with only 8 out of 51 pathway modules being completely conserved. Enzyme comparison based on topology of metabolic modules uncovered diversification between parasite and host that can potentially guide therapeutic intervention. Gene expression data from 4 nematode species were used to study metabolic dynamics over their life cycles. We report unexpected differential metabolism between immature and mature microfilariae of the human filarial parasite Brugia malayi. A set of genes potentially important for parasitism is also reported, based on an analysis of

  17. Pan-phylum Comparison of Nematode Metabolic Potential.

    Science.gov (United States)

    Tyagi, Rahul; Rosa, Bruce A; Lewis, Warren G; Mitreva, Makedonka

    2015-05-01

    Nematodes are among the most important causative pathogens of neglected tropical diseases. The increased availability of genomic and transcriptomic data for many understudied nematode species provides a great opportunity to investigate different aspects of their biology. Increasingly, metabolic potential of pathogens is recognized as a critical determinant governing their development, growth and pathogenicity. Comparing metabolic potential among species with distinct trophic ecologies can provide insights on overall biology or molecular adaptations. Furthermore, ascertaining gene expression at pathway level can help in understanding metabolic dynamics over development. Comparison of biochemical pathways (or subpathways, i.e. pathway modules) among related species can also retrospectively indicate potential mistakes in gene-calling and functional annotation. We show with numerous illustrative case studies that comparisons at the level of pathway modules have the potential to uncover biological insights while remaining computationally tractable. Here, we reconstruct and compare metabolic modules found in the deduced proteomes of 13 nematodes and 10 non-nematode species (including hosts of the parasitic nematode species). We observed that the metabolic potential is, in general, concomitant with phylogenetic and/or ecological similarity. Varied metabolic strategies are required among the nematodes, with only 8 out of 51 pathway modules being completely conserved. Enzyme comparison based on topology of metabolic modules uncovered diversification between parasite and host that can potentially guide therapeutic intervention. Gene expression data from 4 nematode species were used to study metabolic dynamics over their life cycles. We report unexpected differential metabolism between immature and mature microfilariae of the human filarial parasite Brugia malayi. A set of genes potentially important for parasitism is also reported, based on an analysis of gene expression in

  18. Diversifying selection and host adaptation in two endosymbiont genomes

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    Slatko Barton

    2007-04-01

    Full Text Available Abstract Background The endosymbiont Wolbachia pipientis infects a broad range of arthropod and filarial nematode hosts. These diverse associations form an attractive model for understanding host:symbiont coevolution. Wolbachia's ubiquity and ability to dramatically alter host reproductive biology also form the foundation of research strategies aimed at controlling insect pests and vector-borne disease. The Wolbachia strains that infect nematodes are phylogenetically distinct, strictly vertically transmitted, and required by their hosts for growth and reproduction. Insects in contrast form more fluid associations with Wolbachia. In these taxa, host populations are most often polymorphic for infection, horizontal transmission occurs between distantly related hosts, and direct fitness effects on hosts are mild. Despite extensive interest in the Wolbachia system for many years, relatively little is known about the molecular mechanisms that mediate its varied interactions with different hosts. We have compared the genomes of the Wolbachia that infect Drosophila melanogaster, wMel and the nematode Brugia malayi, wBm to that of an outgroup Anaplasma marginale to identify genes that have experienced diversifying selection in the Wolbachia lineages. The goal of the study was to identify likely molecular mechanisms of the symbiosis and to understand the nature of the diverse association across different hosts. Results The prevalence of selection was far greater in wMel than wBm. Genes contributing to DNA metabolism, cofactor biosynthesis, and secretion were positively selected in both lineages. In wMel there was a greater emphasis on DNA repair, cell division, protein stability, and cell envelope synthesis. Conclusion Secretion pathways and outer surface protein encoding genes are highly affected by selection in keeping with host:parasite theory. If evidence of selection on various cofactor molecules reflects possible provisioning, then both insect as

  19. Novel microfilaricidal activity of nanosilver

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    Singh SK

    2012-02-01

    Full Text Available Sunil K Singh1, Kalyan Goswami2, Richa D Sharma2, Maryada VR Reddy2, Debabrata Dash11Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 2Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, IndiaPurpose: The currently available drug repertoire against lymphatic filariasis, a major health hazard in the developing world, is inadequate and is fraught with serious limitations. Thus, the development of an effective antifilarial strategy has become a global research thrust mandated by the World Health Organization. Nanoparticles of silver endowed with antibacterial potency are known to induce apoptosis in eukaryotic cells. The present study was designed to investigate the possible microfilaricidal efficacy of silver nanoparticles and to establish the validity of apoptotic rationale in antifilarial drug designing.Methods: This report analyzed the effect of nanoparticles of silver as well as gold (size range: 10–15 nm on the microfilariae of Brugia malayi obtained from the lavage of peritoneal cavities of infected jirds (Meriones unguiculatus. The study included a microfilarial motility assay, a trypan blue exclusion test, a poly(adenosine diphosphate-ribose polymerase activity study, ethidium bromide/acridine orange differential staining, and transmission, as well as scanning electron microscopic evaluation of ultrastructural changes in microfilariae.Results: The study demonstrates that nanoparticles of silver, but not of gold, elicited significant loss in microfilarial motility. Differential staining of parasites with ethidium bromide and acridine orange, poly(adenosine diphosphate-ribose polymerase activity in microfilarial lysate, and electron microscopic findings underscored apoptotic death of parasites attributable to nanosilver. In a trypan blue exclusion test, the 50% lethal dose of nanosilver was measured to be 101.2 µM, which was higher than the recorded complete

  20. Subcutaneously Administered Ultrafine PLGA Nanoparticles Containing Doxycycline Hydrochloride Target Lymphatic Filarial Parasites.

    Science.gov (United States)

    Singh, Yuvraj; Srinivas, Adepu; Gangwar, Mamta; Meher, Jaya Gopal; Misra-Bhattacharya, Shailja; Chourasia, Manish K

    2016-06-06

    Systemic chemotherapeutic targeting of filarial parasites is unfocused due to their deep seated location in lymphatic vessels. This warrants a prolonged dosing regimen in high doses for an anthelmintic like doxycycline hydrochloride (DOX). In order to provide an alternative, we have constructed ultrafine PLGA nanoparticles of DOX (DPNPs), so as to exploit the peculiarity of lymphatic vasculature underneath the subcutaneous layer of skin, which preferentially allows entry of only 10-100 nm sized particles. DPNPs were constructed using a novel solvent diffusion method aided by probe sonication, which resulted in an average size 95.43 ± 0.8 nm as per DLS, PDI 0.168 ± 0.03, zeta potential -7.38 ± 0.32, entrapment efficiency 75.58 ± 1.94%, and refrigerator stability of 7 days with respect to size in the optimized batch. TEM further substantiated the spherical shape of DPNPs along with their actual nonhydrated size as being well below 100 nm. FTIR analysis of DOX, dummy nanoparticles, and freeze-dried DPNPs revealed that the formulation step did not induce prominent changes in the chemical nature of DOX. The drug release was significantly altered (p < 0.05) with 64.6 ± 1.67% release in 48 h from DPNPs and was dictated by Fickian diffusion. Pharmacokinetic studies in Wistar rats further revealed that DPNPs caused a 16-fold prolongation in attainment of plasma Tmax and a 2-fold extension of elimination half-life (28.569 ± 1.27 h) at a dose of 5 mg/kg when compared to native drug (DOX solution) of the same strength. Contrastingly the trend was reversed in regional lymph nodes where Cmax for DPNPs (820 ± 84 ng/mg) was 4-fold greater, and lymphatic Tmax was attained in one-fourth of what was required for DOX solution. This size based preferential lymphatic targeting resulted in significantly greater in vivo antifilarial activity of DPNPs when compared to DOX solution as gauged by several parameters in Brugia malayi infected Mastomys coucha. Interestingly, the

  1. IMPORTANT NEMATODE INFECTIONS IN INDONESIA

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    Sri Oemijati

    2012-09-01

    Full Text Available At least 13 species of intestinal nematodes and 4 species of blood and tissue nematodes have been reported infecting man in Indonesia. Five species of intestinal nematodes are very common and highly prevalent, especially in the rural areas and slums of the big cities. Those species are Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale, Trichuris trichiura and Oxyuris vermicularis, while Strongyloides stercoralis is disappearing. The prevalence of the soil transmitted helminths differs from place to place, depending on many factors such as the type of soil, human behaviour etc. Three species of lymph dwelling filarial worms are known to be endemic, the urban Wuchereria bancrofti is low endemic in Jakarta and a few other cities along the north coast of Java, with Culex incriminated as vector, high endemicity is found in Irian Jaya, where Anopheline mosquitoes act as vectors. Brugia malayi is widely distributed and is still highly endemic in many areas. The zoonotic type is mainly endemic in swampy areas, and has many species of Mansonia mosquitoes as vectors. B.timori so far has been found only in the south eastern part of the archipelago and has Anopheles barbirostris as vector. Human infections with animal parasites have been diagnosed properly only when adult stages were found either in autopsies or removed tissues. Cases of infections with A. caninum, A.braziliense, A.ceylanicum, Trichostrongylus colubriformis, T.axei and Oesophagostomum apiostomum have been desribed from autopsies, while infections with Gnathostoma spiningerum have been reported from removed tissues. Infections with the larval stages such as VLM, eosinophylic meningitis, occult filanasis and other could only be suspected, since the diagnosis was extremely difficult and based on the finding and identification of the parasite. Many cases of creeping eruption which might be caused by the larval stages of A.caninum and A.braziliense and Strongyloides stercoralis

  2. Genome-wide survey and analysis of microsatellites in nematodes, with a focus on the plant-parasitic species Meloidogyne incognita

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    Guillemaud Thomas

    2010-10-01

    Full Text Available Abstract Background Microsatellites are the most popular source of molecular markers for studying population genetic variation in eukaryotes. However, few data are currently available about their genomic distribution and abundance across the phylum Nematoda. The recent completion of the genomes of several nematode species, including Meloidogyne incognita, a major agricultural pest worldwide, now opens the way for a comparative survey and analysis of microsatellites in these organisms. Results Using MsatFinder, the total numbers of 1-6 bp perfect microsatellites detected in the complete genomes of five nematode species (Brugia malayi, Caenorhabditis elegans, M. hapla, M. incognita, Pristionchus pacificus ranged from 2,842 to 61,547, and covered from 0.09 to 1.20% of the nematode genomes. Under our search criteria, the most common repeat motifs for each length class varied according to the different nematode species considered, with no obvious relation to the AT-richness of their genomes. Overall, (ATn, (AGn and (CTn were the three most frequent dinucleotide microsatellite motifs found in the five genomes considered. Except for two motifs in P. pacificus, all the most frequent trinucleotide motifs were AT-rich, with (AATn and (ATTn being the only common to the five nematode species. A particular attention was paid to the microsatellite content of the plant-parasitic species M. incognita. In this species, a repertoire of 4,880 microsatellite loci was identified, from which 2,183 appeared suitable to design markers for population genetic studies. Interestingly, 1,094 microsatellites were identified in 801 predicted protein-coding regions, 99% of them being trinucleotides. When compared against the InterPro domain database, 497 of these CDS were successfully annotated, and further assigned to Gene Ontology terms. Conclusions Contrasted patterns of microsatellite abundance and diversity were characterized in five nematode genomes, even in the case of

  3. Evolution of hedgehog and hedgehog-related genes, their origin from Hog proteins in ancestral eukaryotes and discovery of a novel Hint motif

    Directory of Open Access Journals (Sweden)

    Bürglin Thomas R

    2008-03-01

    Full Text Available Abstract Background The Hedgehog (Hh signaling pathway plays important roles in human and animal development as well as in carcinogenesis. Hh molecules have been found in both protostomes and deuterostomes, but curiously the nematode Caenorhabditis elegans lacks a bona-fide Hh. Instead a series of Hh-related proteins are found, which share the Hint/Hog domain with Hh, but have distinct N-termini. Results We performed extensive genome searches of the cnidarian Nematostella vectensis and several nematodes to gain further insights into Hh evolution. We found six genes in N. vectensis with a relationship to Hh: two Hh genes, one gene with a Hh N-terminal domain fused to a Willebrand factor type A domain (VWA, and three genes containing Hint/Hog domains with distinct novel N-termini. In the nematode Brugia malayi we find the same types of hh-related genes as in C. elegans. In the more distantly related Enoplea nematodes Xiphinema and Trichinella spiralis we find a bona-fide Hh. In addition, T. spiralis also has a quahog gene like C. elegans, and there are several additional hh-related genes, some of which have secreted N-terminal domains of only 15 to 25 residues. Examination of other Hh pathway components revealed that T. spiralis - like C. elegans - lacks some of these components. Extending our search to all eukaryotes, we recovered genes containing a Hog domain similar to Hh from many different groups of protists. In addition, we identified a novel Hint gene family present in many eukaryote groups that encodes a VWA domain fused to a distinct Hint domain we call Vint. Further members of a poorly characterized Hint family were also retrieved from bacteria. Conclusion In Cnidaria and nematodes the evolution of hh genes occurred in parallel to the evolution of other genes that contain a Hog domain but have different N-termini. The fact that Hog genes comprising a secreted N-terminus and a Hog domain are also found in many protists suggests that this

  4. 半胱氨酸蛋白酶抑制剂的系统发生分析%Phylogenetic Analysis of Cystatin

    Institute of Scientific and Technical Information of China (English)

    李凤梅; 盖雪梅

    2010-01-01

    [目的]对已知半胱氨酸蛋白酶抑制剂基因编码蛋白的分子量、等电点、信号肽、结构域等进行分析.[方法]在NCBI中检索半胱氨酸蛋白酶抑制剂基因,下载相应的氨基酸序列.采用SMART软件预测结构域,用SingalP程序查找信号肽,用TMHMM程序搜寻预测跨膜区.多序列比对采用CLUSTAL W程序.运用MEGA3.1软件,采用Neighbor-joining 法构建进化树.[结果]半胱氨酸蛋白酶抑制A(Homo sapiens)、半胱氨酸蛋白酶抑制M(H. sapiens)、半胱氨酸蛋白酶抑制F(H. sapiens)、半胱氨酸蛋白酶抑制(Mus musculus)、半胱氨酸蛋白酶抑制C(M. musculus)、半胱氨酸蛋白酶抑制F(Rattus norvegicus)、半胱氨酸蛋白酶抑制C(R. norvegicus)、半胱氨酸蛋白酶抑制S(R. norvegicus)、半胱氨酸蛋白酶抑制I(Zea mays)、半胱氨酸蛋白酶抑制(Brugia malayi)、半胱氨酸蛋白酶抑制(Onchocerca volvulus)和半胱氨酸蛋白酶抑制(Acanthocheilonema viteae)有信号肽,其余的半胱氨酸蛋白酶抑制基因没有信号肽,TMHMM程序搜寻结果显示,这些半胱氨酸蛋白酶抑制都没有跨膜区,均为胞外蛋白.SMART软件分析结果表明它们均含有1个高度保守的半胱氨酸蛋白酶抑制剂结构域.多序列比对结果表明半胱氨酸蛋白酶抑制剂基因存在高度保守的QxVxG基序,意味着该基序可能对半胱氨酸蛋白酶抑制剂的抑制活性具有重要意义.系统进化分析可预示半胱氨酸蛋白酶抑制半胱氨酸蛋白酶活性在进化过程中可能也是保守的.[结论]该研究可为半胱氨酸蛋白酶抑制剂抑制半胱氨酸蛋白酶的功能研究方面提供理论参考.