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PubMed1996

A zinc finger gene from Onchocerca volvulus encodes a protein with a functional signal peptide and an unusual Ser-His finger motif.

The filarial parasite Onchocerca volvulus is the causative agent of river blindness. In order to identify genes potentially involved in parasite development we cloned a zinc finger-encoding gene from this species. The ovzf-1 gene represents one member of a family of related zinc finger genes. The predicted ovzf-1 translation product of 447 amino acids includes a hydrophobic signal peptide, which is followed by 13 contiguous finger motifs. The domains of fingers II-XIII display several conserved amino acids and a typical Krüppel-like Cys2-His2 motif. The first finger domain has the two conserved Cys residues replaced with Ser residues; however, it includes all additional amino acids typical of zinc finger domains. The N-terminal domain functions as a signal peptide, as it directs secretion of a reporter protein and a truncated Ovzf protein. Expression of an Ovzf protein via the secretory pathway was also confirmed by demonstrating attachment of N-linked carbohydrates to the recombinant protein. Although the recombinant Ovzf protein also includes a signal peptide, immunofluorescence analyses localize it inside a specific compartment of the infected insect cell. Expression of ovzf mRNA is developmentally regulated; no specific transcript is detected in adult female worms but in the infective L3. Identification of a secreted protein that might function in modulating gene expression of host cells provides an interesting tool for the study of parasite-host interaction on a biochemical and molecular level.

Amino Acid Sequence↗
PubMed1996

Molecular cloning, expression, and localization of E1, an Onchocerca volvulus antigen with similarity to brain ankyrin.

Protective immunity against human onchocerciasis may best be reflected by the existence of individuals who in spite of exposure to the filarial nematode Onchocerca volvulus do not develop disease (putatively immune). We observed preferential recognition of an O. volvulus antigen of approximately 90 kDa by sera from putatively immune individuals compared with sera from diseased individuals. Screening of an adult worm cDNA library with one serum recognizing this antigen almost exclusively led to the identification of a full length clone of 2043 base pairs designated E1. The open reading frame of 462 amino acid residues shows similarity to human brain ankyrin. E1 appears to represent a small transcript of the O. volvulus ankyrin gene. The nonfusion protein obtained by expression of the complete E1 cDNA exhibits an apparent molecular mass of 90 kDa on SDS-polyacrylamide gel electrophoresis. An antiserum against the recombinant protein reacts with the 90-kDa antigen in O. volvulus extract. In O. volvulus, E1 was localized in the neuronal cell bodies, the nerve ring, and the extracellular clefts of the basal labyrinth. These results identify an ankyrin-related O. volvulus protein as an immunogen to putatively immune individuals, suggesting that neuronal proteins may be important targets for immunity against O. volvulus in vivo.

Amino Acid Sequence↗
PubMed1996

Cloning of a cysteine protease required for the molting of Onchocerca volvulus third stage larvae.

We have investigated the involvement of a cysteine protease in the development of Onchocerca volvulus fourth stage larvae (L4) by testing the effect of cysteine protease inhibitors on the survival of third stage larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluoromethylketones, viability of either L3 or L4 was not affected. However, the inhibitors reduced the number of L3 that molted to L4 in vitro in a time- and dose-dependent manner. Molting was completely inhibited in the presence of 50-250 microM inhibitor. Ultrastructural examination of L3 that did not molt in the presence of inhibitors indicated that new L4 cuticle was synthesized, but there was no separation between the L3 and the L4 cuticles. The endogenous cysteine protease was detected in molting larvae after binding to labeled inhibitors, and by antibodies directed against a recombinant O. volvulus L3 cysteine protease that was cloned and expressed. The enzyme was detected in cuticle regions where the separation between the cuticles occurs in molting larvae. These studies suggest that molting and successful development of L4 depends on the expression and release of a cysteine protease.

Amino Acid Sequence↗
PubMed1997

The Ov20 protein of the parasitic nematode Onchocerca volvulus. A structurally novel class of small helix-rich retinol-binding proteins.

Ov20 is a major antigen of the parasitic nematode Onchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite. DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli. Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-DL-alpha-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol. The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized beta-sheet-rich mammalian retinol- and fatty acid-binding proteins. Secondary structure prediction algorithms indicate that a alpha-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of beta structures, and this was confirmed by circular dichroism. The protein is highly stable in solution, requiring temperatures in excess of 90 degrees C or high denaturant concentrations for unfolding. Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes. The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy.

Amino Acid Sequence↗
PubMed2001

Expression and secretion of a larval-specific chitinase (family 18 glycosyl hydrolase) by the infective stages of the parasitic nematode, Onchocerca volvulus.

A recently reported chitinase gene, expressed in the infective, third-stage (L3) larvae of the human filarial parasite Onchocerca volvulus, belongs to the family 18 glycosyl hydrolases and has been designated Ov-chi-1. The gene product of Ov-chi-1 is chitinolytic. Allosamidin ablates activity of the native enzyme in a dose-dependent manner but did not significantly inhibit the moulting of L3 larvae. Mono-specific antibodies were used to characterize Ov-CHI-1 as a 60-kDa protein expressed almost exclusively in L3 stages. Immunoelectron microscopy showed that Ov-CHI-1 expression is initiated in late L2 larvae and increases markedly in infective, L3 larvae. It is synthesized exclusively in the glandular esophagus and stored within discrete secretory granules. Secretion occurs through de-granulation during post-infective development, and the primary route of transport appears to be via the pseudo-coelom. An orthologue of Ov-chi-1 was detected in Caenorhabditis elegans by BLAST analysis. It is constitutively expressed at a low level and is overexpressed in dauer larvae and embryonated eggs. It is chitinolytic. We conclude that Ov-CHI-1 is a highly stage-specific enzyme that may have a role in infectivity of the parasite, aiding escape from the vector or participating in early post-infective migration and/or development. The identification of an orthologue in C. elegans opens the way for further studies into the biological function(s) of this intriguing parasite product.

Amino Acid Sequence↗
PubMed2003

Cloning and biochemical characterization of blisterase, a subtilisin-like convertase from the filarial parasite, Onchocerca volvulus.

Blisterase is a subtilisin-like proprotein convertase of nematodes. The enzyme is named after the blistered cuticle found in Caenorhabditis elegans with the bli-4 e937 mutation. The critical role of the enzyme in cuticle production makes it a potential drug target for parasitic nematodes. We have cloned and expressed blisterase from the parasitic nematode Onchocerca volvulus, a major cause of blindness in Africa. The catalytic domain of the protease exhibits 84% identity with the corresponding domain of its closest homologue, C. elegans blisterase. O. volvulus blisterase expressed in insect cells has maximal activity in 1 mm calcium at neutral pH. The protease is inhibited by EDTA, the suicide substrate decanoyl-RVKR-chloromethylketone, alpha1-antitrypsin Portland and by its own propeptide. Substrate assays with fluorescent peptides show that O. volvulus blisterase requires a P4 arginine and a basic amino acid at P1 for cleavage. The kcat of blisterase on the peptide substrate, t-butyloxycarbonyl-RVRR-4-methylcoumaryl-7-amide was determined to be 0.018 s-1. In vitro cleavage studies with the nematode polyprotein antigen demonstrated that blisterase cleaved at tetrabasic (RRKR) but not at dibasic (KR) sites. This report describes the first biochemical characterization of the nematode specific protease, blisterase.

Amino Acid Chloromethyl Ketones↗
PubMed2005

Structure of the major cytosolic glutathione S-transferase from the parasitic nematode Onchocerca volvulus.

Onchocerciasis is a debilitating parasitic disease caused by the filarial worm Onchocerca volvulus. Similar to other helminth parasites, O. volvulus is capable of evading the host's immune responses by a variety of defense mechanisms, including the detoxification activities of the glutathione S-transferases (GSTs). Additionally, in response to drug treatment, helminth GSTs are highly up-regulated, making them tempting targets both for chemotherapy and for vaccine development. We analyzed the three-dimensional x-ray structure of the major cytosolic GST from O. volvulus (Ov-GST2) in complex with its natural substrate glutathione and its competitive inhibitor S-hexylglutathione at 1.5 and 1.8 angstrom resolution, respectively. From the perspective of the biochemical classification, the Ov-GST2 seems to be related to pi-class GSTs. However, in comparison to other pi-class GSTs, in particular to the host's counterpart, the Ov-GST2 reveals significant and unusual differences in the sequence and overall structure. Major differences can be found in helix alpha-2, an important region for substrate recognition. Moreover, the binding site for the electrophilic co-substrate is spatially increased and more solvent-accessible. These structural alterations are responsible for different substrate specificities and will form the basis of parasite-specific structure-based drug design investigations.

Amino Acid Sequence↗
PubMed2005

Characterization of a novel filarial serine protease inhibitor, Ov-SPI-1, from Onchocerca volvulus, with potential multifunctional roles during development of the parasite.

A novel filarial serine protease inhibitor (SPI) from the human parasitic nematode Onchocerca volvulus, Ov-SPI-1, was identified through the analysis of a molting third-stage larvae expressed sequence tag dataset. Subsequent analysis of the expressed sequence tag datasets of O. volvulus and other filariae identified four other members of this family. These proteins are related to the low molecular weight SPIs originally isolated from Ascaris suum where they are believed to protect the parasite from host intestinal proteases. The two Ov-spi transcripts are up-regulated in the molting larvae and adult stages of the development of the parasite. Recombinant Ov-SPI-1 is an active inhibitor of serine proteases, specifically elastase, chymotrypsin, and cathepsin G. Immunolocalization of the Ov-SPI proteins demonstrates that the endogenous proteins are localized to the basal layer of the cuticle of third-stage, molting third-stage, and fourth-stage larvae, the body channels and multivesicular bodies of third-stage larvae and the processed material found between the two cuticles during molting. In O. volvulus adult worms the Ov-SPI proteins are localized to the sperm and to eggshells surrounding the developing embryos. RNA interference targeting the Ov-spi genes resulted in the specific knockdown of the transcript levels of both Ov-spi-1 and Ov-spi-2, a loss of native proteins, and a significant reduction in both molting and viability of third-stage larvae. We suggest the Ov-SPI proteins play a vital role in nematode molting by controlling the activity of an endogenous serine protease(s). The localization data in adults also indicate that these inhibitors may be involved in other processes such as embryogenesis and spermatogenesis.

Amino Acid Sequence↗
PubMed2001

The prevalence, intensity and clinical manifestations of Onchocerca volvulus infection in Toro local government area of Bauchi State, Nigeria.

Between January and October 1994, a study of the prevalence, intensity and clinical manifestations of onchocerciasis in nine communities of Toro local government area of Bauchi State, Nigeria was undertaken using the skin-snip method. Of the 1117 inhabitants examined, 188 (16.8%) were positive for microfilariae of Onchocerca volvulus. The prevalence of onchocerciasis was significantly higher (P < 0.05) among males than females, in subjects 21 years of age and above than in those in the first two decades of life, in nomads, farmers, hunters and fishermen than smiths and traders. Intensity of infection was light, not exceeding a geometric mean of 5.3 microfilaria per 2 mm skin bite. Preponderance of positive cases below 20 years presented no chronic signs. Conversely, persons above 20 years had higher microfilaria counts which coincides with the period when most clinical signs manifest. Microfilarial-rate and -density in relation to age were closely associated (r = 0.75, P < 0.001). The need for a sustained mass distribution of Mectizan in these communities is highlighted.

Adolescent↗
PubMed2001

Studies on the macrofilarial population of Onchocerca volvulus in hyper-endemic villages of the Central province of Cameroon.

The population structure of Onchocerca volvulus macrofilariae was studied in villages of central Cameroon where onchocerciasis is hyper-endemic. One nodule selected at random was removed from each of 576 adult males, and examined by histology. The numbers of male and female worms/nodule, and the status of the female worms (fecund, non-fecund, or dead) were recorded. The observations were analysed to evaluate whether the mean numbers of worms of each category varied in relation to the patient's age, the level of endemicity in his village, the anatomical localization of the nodule, the weight of the nodule, and the total number of palpable nodules harboured by the patient. The results obtained were very similar to those reported from West Africa. The mean numbers of dead female worms/nodule were relatively high in the villages with the lowest levels of endemicity. The mean numbers of fecund females and of live males were significantly higher in the nodules located around the knees. These results provide information which might be useful in modelling the population dynamics of O. volvulus, and also in the context of trials of any potentially macrofilaricidal drugs.

Adolescent↗
PubMed2001

Misidentification of Onchocerca volvulus as guinea worm.

Over the past 10 years, the status of human infection with guinea worm (Dracunculus medinensis) in the Central African Republic (CAR) has been difficult to ascertain. It is unclear if indigenous cases are occurring and whether cases are migrating into the CAR from surrounding countries. A team of investigators visited the CAR in July-August 2000, to attempt to ascertain the presence of indigenous transmission. No cases of true guinea-worm infection (i.e. dracunculiasis) were detected, but three cases of human infection with Onchocerca volvulus, each of which had been misidentified as dracunculiasis, were detected. The unusual presentation of skin blisters and extraction of an intact female O. volvulus are described. As a result of this investigation, and the confusion of onchocerciasis being misidentified as dracunculiasis, the presence of endemic transmission of guinea worm in the CAR remains in question.

Adult↗
PubMed1979

The development of Onchocerca volvulus in two temperate blackfly species, Simulium ornatum Meigen and Simulium lineatum Meigen.

In view of the promise shown by temperate simuliids for laboratory colonization, the ability of Simulium ornatum and S. lineatum to support the development of Onchocerca volvulus was investigated. An injection technique is described for the introduction of O. volvulus microfilariae from a chimpanzee into female simuliids in the absence of a bloodmeal. RPMI 1640 medium, with penicillin, streptomycin and foetal calf serum, proved satisfactory as an inoculation medium. Third stage larvae were obtained in both S. ornatum and S. lineatum after seven days at 27 degrees C. The rate of development of the parasite in S. ornatum is recorded.

Animals↗
PubMed1987

Studies on the dynamics of transmission of onchocerciasis in a Sudan-savanna area of North Cameroon III. Infection rates of the Simulium vectors and Onchocerca volvulus transmission potentials.

Populations of Simulium damnosum s.str. and S. sirbanum were examined for infections with filarial parasites during three years in the areas of Tcholliré and Touboro, at sites at different distances from Simulium breeding rivers, and in relation to villages with different endemicities of onchocerciasis. A total of 60,353 flies from 23 fly-catching sites were dissected. The overall infection rate was low, 11.8% of 35,357 parous flies dissected. 1681 flies (4.8% of the total parous) contained 3557 infective larvae, 68.8% of which were morphologically indistinguishable from Onchocerca volvulus and 31.2% were infective 'Type D' larvae of non-human origin, indicating a high degree of zoophily of the fly populations. It was estimated that only 20-40% of all bloodmeals were taken from man. The majority (54%) of all infective O. volvulus larvae were found in the heads of the flies, the remainder being in the thorax (34%) and abdomen (12%). Only 54% of the O. volvulus infective larvae left their vectors during a bloodmeal which, however, was not completed in most cases. During the rainy season infection rates with O. volvulus infective larvae were 3.5% of the total parous flies, as compared with 1.8% during the dry season. The average number of infective larvae of O. volvulus per infective fly was 2.6 and 2.2 during the rainy and dry seasons respectively. These variations in the vectorial efficiency of the fly populations, as well as variations from one site to another, could be explained by different survival rates and man-biting habits of the various vector populations during the dry and rainy seasons and in different regions, rather than by different endemic profiles of onchocerciasis in the human population. The intensity of transmission varied seasonally and was highest (1609 to 3076 infective larvae/man/year) near the main breeding sites, where transmission was almost perennial. At distances of more than 3 km from the river transmission was mainly restricted to the rainy season and the Annual Transmission Potential was below 200, whereas low to zero levels of transmission were measured inside villages more than 3 km distant from the river. The coefficient of variation of the Annual Transmission Potentials over the three years of studies was from 31% to 192% of the mean, being higher that the variations in the corresponding levels of the biting rates, due to the low numbers of infective flies dissected at sites with low transmission.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Observations and reflections on the immature stages of Onchocerca volvulus in the human host.

Knowledge of the morphology, migrations and development rates of the L3, L4 and prepubertal L5 stages of Onchocerca volvulus in the vertebrate host is an essential prerequisite to work vaccine development of chemoprophylaxis. After a brief review of the scanty literature available on this subject a description is given of two immature worms, one male and one female, found in an O. volvulus nodule from Guatemala. The male, which measured 12 mm x 40 micron(s), had fine regular transverse cuticular annulations, two prominent spicules, and an undifferentiated testis. The female, which measured 20 mm x 40 micron(s), had not yet developed cuticular ridges but showed a vulva, gut, and two rudimentary genital tracts. It is thought that these worms may have been six to 12 weeks old. The likely course of the early development of O. volvulus in the vertebrate host is discussed, based on analogy with O. lienalis in cattle; and some of the factors to be taken into account when pursuing the search for O. volvulus immatures are described.

Animals↗
PubMed1992

Inhibition of carbohydrate uptake of Onchocerca volvulus infective larvae (L3) by effector cell constituents.

The in vitro killing of Onchocerca volvulus infective third stage larvae (L3) by components of their human hosts' defence mechanisms is not well documented, as no suitable assay exists. Motility is inappropriate as a criterion of larval viability because of the unsteady winding movements of L3. In the present study, a metabolic parameter for larval viability, the uptake of [3H]2-deoxy-D-glucose, was evaluated. To demonstrate the reproducibility and validity of this test, the oxygen radical hydrogen peroxide (H2O2) was applied to viable L3 and the death of L3 demonstrated by a 90% reduction in glucose uptake. The incorporation of glucose by the filarial larvae was also determined after in vitro exposure to lysates of the putative effector cells, i.e. eosinophilic and neutrophilic granulocytes and monocytes. Effector-cell-derived components led to a 30-80% dose-dependent decrease in deoxy-glucose uptake, with a half-maximal effect at about 500 micrograms ml-1. These experiments demonstrate, for the first time, the deleterious impact of effector cell constituents on the metabolic activity of O. volvulus L3. The assay could be used to evaluate the effect of distinct natural or recombinant effector molecules on the viability of O. volvulus infective larvae and to investigate the effect of parasite molecules which interfere with effector mechanisms.

Animals↗
PubMed1996

Onchocerca volvulus and O. lienalis: the microfilaricidal activity of moxidectin compared with that of ivermectin in vitro and in vivo.

The activity of the veterinary drug moxidectin against Onchocerca volvulus and On. lienalis microfilariae (mf), both in vitro and in experimentally infected CBA/Ca mice, was compared with that of ivermectin. The in-vitro results demonstrated that both compounds (at a concentration of 10(-7) M) significantly reduced the mf motility index (MI) throughout the 7-day culture period and that this reduction was similar for the two compounds. Mice were treated with moxidectin and ivermectin by subcutaneous injection (sc) or orally (po); the two routes were equally efficacious. When mice infected with On. lienalis were treated with one of the drugs at 3.2, 1.6, 0.8, 0.4 or 0.2 micrograms/kg.day on days 3-7 post-infection, with necropsy on day 18, moxidectin cleared more mf than ivermectin at all of the doses examined. In mice treated with a single dose (on day 3 post-infection), 150 or 15 micrograms/kg moxidectin completely cleared the mf whereas 1.5 micrograms/kg produced a 90%-96% reduction in mf recoveries. Following ivermectin treatment at the same doses, mf were virtually cleared at 150 micrograms/kg, with a 98% reduction at 15 micrograms/kg but no significant effect at 1.5 micrograms/kg. When mice with On. volvulus infections were treated with a single dose of moxidectin at 15 or 1.5 micrograms/kg, there were reductions in mf recoveries of 96% and 23%, respectively, compared with only a 48% reduction with 15 micrograms ivermectin/kg and a 2% increase with 1.5 micrograms ivermectin/kg. In order to examine the persistence and activity of each drug, mice were treated with a single dose of 150 micrograms/kg up to 28 days before infection. Moxidectin was found to be more efficacious (with subsequent 99.9% reduction in mf when given 28 days pre-infection and a 100% reduction when give 16 or 4 days before or 3 days after infection) than ivermectin (giving reductions of 57.1%, 66.7%, 100% and 100%, respectively). The further evaluation of moxidectin and its potential usefulness for the treatment of human onchocerciasis are discussed.

Animals↗
PubMed1997

Identification of larval-stage-specific antigens of Onchocerca volvulus uniquely recognized by putative immune sera from humans and vaccination sera from animal models.

Immunity to Onchocerca volvulus is indicated by the existence of putative immune individuals (PI), who do not develop patent infections or clinical symptoms after living in endemic areas for extended periods of time. To gain insight into the nature of the humoral response of PI from Liberia and Ecuador, their sera and those of infected individuals were investigated and compared using western blots. The antigen preparations used were extracts of third- and fourth-stage larvae (L3 and L4, respectively), excretory-secretory proteins (ES-L3) and extracts of third-stage larvae (L3-day2) that had been cultured for 2 days, and extracts of nodular and skin microfilariae. Whereas some antigens were commonly recognized by all individuals, many stage-specific antigens (of 68, 60, 55, 46, 43, 42, 20 and 18 kDa in L3; 100, 88, 80, 50, 48, 45, 40 and 20 kDa in L3-day2; 110, 80 and 72 kDa in ES-L3; 48, 18, 16 and 14 kDa in L4; and 115, 60, 47, 43 and 17 kDa in skin and/or nodular microfilariae) were uniquely recognized by sera from PI. Some of the unique antigens were also recognized by sera from mice and a chimpanzee that were resistant to challenge after immunization with irradiated third-stage larvae. The results support the validity of the assumption that a unique status of immunity develops in certain individuals living in areas where onchocerciasis is endemic.

Animals↗
PubMed1997

Comparison of the use of skin scarification and skin biopsies to determine the prevalence and intensity of Onchocerca volvulus infection.

Two methods for the detection of Onchocerca volvulus microfilariae in the skin were compared in a population-based survey in Gatete, Murama and Jerama, three 'collines' (administrative units) in Burundi. Of the 513 subjects examined, each by both methods, 131 (25.5%) were found positive by skin scarification and 119 (23.2%) by membrane filtration of the fluid in which two skin snips had been incubated. Whilst the sensitivity of the two methods was similar (90% for scarifications and 82% for skin snips), more microfilariae were found by scarification than by biopsy (with geometric mean numbers of 9.1 and 4.1, respectively). Also, more of the cases with infections of low intensity (i.e. < or = 5 microfilariae detected) were found by scarification than by skin snipping (77.9% v. 60.3%; P = 0.06). The endemicity of onchocerciasis and age- and sex-specific prevalences and intensities of infection could be determined equally well using either method. The shortcomings of past comparisons between scarification and skin-snip methods are discussed. Skin scarification has many advantages for the detection of infected individuals in rural health centres and for use in population-based surveys. It may also be helpful when mixed filarial infections are present (especially where onchocerciasis and loiasis co-occur).

Adolescent↗