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PubMed1993

The Onchocerca volvulus mRNAs for a hsp70, a collagen-like protein and a ribosomal protein possess a 5' spliced leader sequence.

To detect and analyse Onchocerca volvulus mRNAs possessing a spliced leader (SL) sequence, a SL cDNA library was constructed from O. volvulus RNA using a PCR method. Three of the sixty cDNAs analysed displayed significant homology to a known sequence. These cDNAs encode a hsp70, a collagen-like protein and a 60S ribosomal protein. The remaining cDNAs analysed either encoded proteins with no apparent homology to known sequences or they appeared to be generated from RNAs with an internal SL and did not possess an open reading frame.

Amino Acid Sequence↗
PubMed1994

IL-4- and IL-5-dependent protective immunity to Onchocerca volvulus infective larvae in BALB/cBYJ mice.

A significant reduction in challenge worm survival occurred when BALB/cBYJ mice were vaccinated against Onchocerca volvulus infective third stage larvae (L3) by using irradiated O. volvulus L3. Challenge infections consisted of L3 implanted in diffusion chambers, which were used as a means to contain, and thus efficiently recover, the larvae from the host. The goal of the present study was to describe the mechanism of immune-mediated killing of O. volvulus L3 in diffusion chambers in mice. Direct contact between host cells and parasites was required for killing of larvae in immunized hosts. To define the mechanism of immune-mediated killing in this system, the time of influx of cells and cytokines into the infection site was compared with the time challenge infections were killed. The only cell type that was found to increase in diffusion chambers in immunized mice was eosinophils; maximal levels of eosinophils were coincident with the time of parasite killing. IL-5 was found in diffusion chambers of immunized mice coincident with the time of parasite killing; IL-5 was not found in diffusion chambers recovered from control mice. Significant levels of IFN-gamma were absent in the diffusion chambers of both groups. Immunized mice were treated with mAb to eliminate IL-5 or IL-4 to assess the role these cytokines or their by-products play in larval killing. Elimination of either IL-5 or IL-4 significantly reduced the protective effects of vaccination against larval O. volvulus.

Animals↗
PubMed1993

Identification of a zinc finger encoding gene in Onchocerca volvulus.

Eukaryotic transcription factors can be identified and classified according to conserved DNA-binding motifs and conserved regulatory domains. The functional and structural analysis, and the conservation of these genes among metazoans emphasizes the importance of these DNA-binding proteins in development and differentiation. In order to identify genes with common DNA-binding motifs in the genome of Onchocerca volvulus we have cloned a zinc finger encoding gene of the structure C2-H2. Using the Caenorhabditis elegans sex determining tra-1 gene as a probe we have identified two tra-1 related genes in the genome of O. volvulus. The zinc finger region of one of these genes (OvZf1) was subcloned and sequenced. The predicted protein has at least eight consecutive zinc fingers and each finger possesses the characteristic paired cysteine- and histidine residues and the proper spacing of the amino acids between the conserved residues.

Amino Acid Sequence↗
PubMed1993

Absence of sperm from the seminal receptacle of female Onchocerca volvulus following multiple doses of ivermectin.

It is already known that multiple doses of ivermectin have a profound effect on embryonic development in Onchocerca volvulus and that this appears to operate mainly at the single cell stage. To investigate this further, we examined adult female O. volvulus originating from patients treated either with placebo or one, four or five doses of ivermectin. The reproductive organs were dissected out of the worm and examined for evidence of oogenesis and for the quantity of sperm and oocytes in the seminal receptacles. A single dose of ivermectin had no measurable effect on oogenesis or on the amount of sperm and oocytes compared to placebo. However after multiple doses of ivermectin a significantly lower proportion of seminal receptacles contained sperm and there was a significantly higher proportion of ovaries with impaired oogenesis compared to placebo. It is concluded that the reduction in the number of multicellular embryonic stages from worms exposed to multiple doses of ivermectin is due, at least in part, to a major reduction in the effective insemination of female worms and to a minor impairment of oogenesis.

Animals↗
PubMed1993

Survival and development of larval Onchocerca volvulus in diffusion chambers implanted in primate and rodent hosts.

Third-stage larvae (L3) of Onchocerca volvulus were implanted in diffusion chambers in chimpanzees, mangabey monkeys, rhesus monkeys, squirrel monkeys, and inbred strains of mice, jirds, and rats for 3-63 days. At different times during the experimental period, larvae were recovered and assessed for their viability and development. Survival and growth rates were equal regardless of whether the implanted larvae were fresh or cryopreserved. Survival and growth rates of the larvae did not differ among the primate and rodent hosts tested, with the exception of squirrel monkeys and rats, which were resistant to infection. Molting from L3 to fourth-stage larvae began on day 3 and continued through day 14 in the primates and rodents. The primate and rodent models developed in the present study will be useful for the study of the immunology and chemotherapy of onchocerciasis.

Animals↗
PubMed1993

The population dynamics of Onchocerca volvulus in the human host.

The quantitative aspects of onchocerciasis that focus on the relationship between the number of adult female Onchocerca volvulus in the human host, the total numbers of microfilariae (mfs) in the body at any one time, and the numbers of infective larvae (L3) to which a person is exposed over time, have received little attention. This paper attempts to investigate the problem from three starting points:--(a) using the numbers of palpable nodules to estimate the numbers of productive female worms in the body; (b) using the concentrations of mfs in skin snips to estimate the total load of mfs in the body; and (c) using the Annual Transmission Potential to estimate the numbers of L3 developing to productive female maturity. By analysing published and unpublished data relating to the forest and savanna zones of West Africa, these three approaches indicate that a relatively large proportion of adult female worms lie deep in the body and are impalpable from the surface. They also provide numerical results, of the same order to magnitude, which can be linked to provide estimates of the total numbers of adult female worms and mfs in the bodies of persons with infections of different intensities.

Africa, Western↗
PubMed1993

Cryopreservation of infective larvae of Onchocerca volvulus (Filarioidea: Onchocercidae).

Infective larvae (L3) of Onchocerca volvulus were procured in Liberia, West Africa, in the natural black fly vector, Simulium yahense. A cryobiological technique was developed to preserve L3 of O. volvulus that were fully viable after thawing. Larvae were treated before cooling with 4 cryoprotective compounds. Three compounds, dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol, were prepared with distilled water. The fourth compound was DMSO prepared in different concentrations with 0.25 M sucrose. The treatment with DMSO + 0.25 M sucrose cryoprotectant resulted in the highest survival of infective larvae. Five cooling rates between 0.5 C/min and 20.0 C/min were applied. The highest survival of L3 was with the cooling rate of 1.0 C/min. Two-step cooling of L3 was applied. In the first step, L3's were frozen to 5 levels from -10.0 C to -20.0 C, -30.0 C, -40.0 C, -60 C, and -80.0 C, and in the second step, larvae were transferred into liquid nitrogen at -196 C for rapid cooling and storage. The survival was the highest when larvae were cooled to approximately -40 C prior to transfer into liquid nitrogen. Slow, gradual, and rapid thawing procedures were applied. The survival was the highest in rapid warming.

Animals↗
PubMed1978

Histochemical enzyme-staining patterns of onchocerca volvulus microfilariae and their occurrence in different onchocerciasis areas.

Histochemical differentiation of Onchocerca volvulus microfilariae from 164 patients in West African rain-forest (Liberia), Sudan-savanna (Upper Volta), Guatemala and the Yemen has been carried out using a staining method for the demonstration of acid phosphatase. Intrauterine microfilariae showed considerable changes in their enzyme activity during embryonic development which are probably associated with the maturation of the parasite before migration to the tissues. Five distinct types of staining patterns could be distinguished among microfilariae from the skin according to the localization of the enzyme in specific structures of the microfilaria. Two or more types of staining patterns were found in most persons in the different geographic regions. There were significant differences in the overall distribution of the various staining patterns in persons from the different areas. At the present state of our knowledge, little is known about the nature and significance of these differences in the staining patterns of microfilariae. The question of whether they can be ascribed to an ageing process, strain differences or other factors is discussed.

Acid Phosphatase↗
PubMed1993

Characterization of a recombinant T cell and B cell reactive polypeptide of Onchocerca volvulus.

To identify potentially protective Ag of the filarial nematode Onchocerca volvulus on the molecular level we screened a cDNA library of O. volvulus with a human serum raised against radiation-attenuated infective larvae of O. volvulus. A cDNA clone of 218 bp (OvL3-1) was selected for further studies. It was expressed in Escherichia coli and affinity purified recombinant polypeptide was tested for its ability to stimulate in vitro PBMC from African onchocerciasis patients and PBMC from chimpanzees experimentally infected with O. volvulus. An enhanced cell proliferation by PBMC was observed in many patients after stimulation with the recombinant OvL3-1 polypeptide. In addition, some patients' PBMC responded to OvL3-1 stimulation with enhanced IL-2 production. Infected chimpanzees also showed an increase in T cell proliferation. Onchocerciasis patients had variable levels of specific antibodies directed to the recombinant polypeptide when sera were tested by ELISA. A mAb directed against the recombinant protein located the native target Ag in the muscles of the adult worm. The molecular mass of native OvL3-1 was found to be 50 kDa on immunoblots. Polymerase chain reaction analysis of RNA from different life stages of the parasite showed that OvL3-1 is transcribed in all parasite stages within the mammalian host. A homologous gene is also present in other filarial parasites. The protein corresponding to OvL3-1, therefore, represents an immunogen present during the whole life-span of the parasite, and because of its B and T cell stimulatory properties, it may be a candidate for a protective Ag in human filariasis.

Adult↗
PubMed1993

Effect of ivermectin treatment on microfilarial load in patients with Onchocerca volvulus in Bebeka, Ethiopia.

In April 1990, one hundred ninety people from Bebeka Coffee Plantation, Kefa administrative region, western Ethiopia, all with moderate to heavy infection with Onchocerca volvulus were treated with ivermectin (150 mcg/kg body wt.). Clinical examination and microfilarial counts in skin snips were repeated at intervals for eight months. Therapy was associated with minimal side effects. Significant difference in mean microfilarial load was observed throughout the eight months post-treatment period. Difference in repopulation of microfilariae between four and eight months was not significant in either sex and all ages. Of all cases, 71 (37.5%) maintained zero microfilarial counts after four months, while only 38 (20%) remained at zero level until the end of eight months. The merit of a yearly treatment programme is discussed.

Adolescent↗
PubMed1995

A putative protein related to human chemokines encoded antisense to the cDNA of an Onchocerca volvulus antigen.

Chronic hyperactive dermatitis (sowda) in humans infected with the filarial parasite Onchocerca volvulus appears to reflect a hyperresponsiveness to parasite antigens. To identify antigens which play a role in this hyperresponsiveness an expression cDNA library of adult O. volvulus was screened with sera from patients with sowda. One further characterized cDNA clone, S1, consisting of 723 bp, surprisingly shows open reading frames (ORF) in both orientations. While a single ORF of 171 amino acids is present in sense orientation, a putative ORF of 95 AA is found in antisense orientation (aS1). Whereas no homologies to known proteins are found in S1, the sequence of aS1 shows a striking structural homology to human CC chemokines. The genomic organization of the coding region of aS1 shows the conserved three exon/two intron structure of the CC chemokine family. In adult worms transcription of mRNA corresponding to S1 but not to aS1 was detected. Expression of S1 as a non fusion protein and Western blot analysis revealed antibody recognition by all sera from patients with sowda, by 60% of sera from patients with the generalized form of onchocerciasis, but not by sera of exposed individuals with no evidence of onchocerciasis. IgG subclass analysis showed that IgG3 reactivity was restricted to sowda sera. In adult worms the S1 protein was localized to the hypodermis. Here we present the cloning and characterization of an O. volvulus antigen, which may be useful in the diagnosis of onchocerciasis. Furthermore, the results suggest the presence of a gene structurally related to human inflammatory cytokines in antisense orientation, raising the question of bidirectional transcription in O. volvulus.

Amino Acid Sequence↗
PubMed1995

Organization and structure of an Onchocerca beta-tubulin gene.

In filarial worms, as in other eukaryotes, microtubules are essential multifunctional components. The major protein of microtubules is tubulin, a heterodimer of two distinct polypeptides, alpha and beta. Tubulin is particularly important in helminthic parasites as a target for anthelminthic benzimidazoles, which bind to it and inhibit microtubule assembly. Two genomic Onchocerca gibsoni libraries were constructed in lambda NM1149 (EcoRI and HindIII). Three clones accounted for the entire gene: one from the EcoRI library (using a Plasmodium falciparum probe) containing the central part of the gene, and two from the HindIII library (using as probes PCR amplified fragments from the ends of the EcoRI clone) which, respectively, contained the 5'- and 3'-ends of the gene. The sequencing procedure for the EcoRI clone relied on the construction of a double-digested DraI/HindIII shotgun library. A number of recombinants were sequenced and aligned with each other for comparison. The sequencing of the overlapping 5'- and 3'-end clones was done by using a series of oligonucleotides. The sequence of the O. gibsoni beta-tubulin gene was completely determined, revealing an exceptionally complex structure as compared to the known beta-tubulin genes: 5,797 base pairs containing 12 exons and 11 introns. The deduced polypeptide is 444 amino acids long, and its sequence is highly conserved. The position of some introns appear to demarcate functional domains within the protein.

Amino Acid Sequence↗
PubMed1996

Integrated control of Onchocerca volvulus infection in a hyperendemic zone in Sierra Leone.

A simplified integrated method of onchocerciasis control involving mass chemotherapy with ivermectin, nodulectomy and health education was carried out in south central Sierra Leone. The prevalence of Onchocerca volvulus in the village community ranged from a low of 7.4% among the International School children to a high of 97.0% in Gbessebu. There was a significant (3-4%) overall reduction in the rate of infection after one year of ivermectin treatment (G = 2565.97, df = 11, P < 0.001). In addition, ivermectin treatment had a significant effect on the college community (G = 1168.21, df = 5, P < 0.001) and from year to year (G = 187.29, df = 1, P < 0.001), though the effect indicates the reduction varied between communities. Mass ivermectin treatment significantly (G = 1 3.79, df = 1, P < 0.005) reduced the rate of infection among school children, while the prevalence and intensity of infection was reduced by nodulectomy. Health education involving the community gave us an entry point and allowed us to gain the maximum cooperation and participation from the communities.

Adolescent↗
PubMed1977

On the development of onchocerca volvulus in mosquitoes.

In an attmept to find an experimental vector for Onchocerca volvulus which can be easily be bred under laboratory conditions, the susceptibility of Anopheles gambiae and Aedes aegypti to infection with O. volvulus was studied. The mosquitoes were fed on a membrane feeding apparatus containing microfilariae of O. volvulus. Whereas A. gambiae was refractory to infection. 14.9% of A. aegypti females became infected with developing stages, 11,2% showed young and mature third-stage larvae as well as some which had developed abnormally. However, no infective larvae of O. volvulus were found in the abdomen or the head of the mosquitoes.

Aedes↗
PubMed1996

Do excretory-secretory products of Onchocerca gibsoni contain phosphorylcholine attached to O-type glycans?

Excretory-secretory products (ES) of adult male Onchocerca gibsoni contain phosphorylcholine (PC). PC-ES are detected as a smear of M(r) approximately 60- approximately 200 kDa by western blotting employing a monoclonal antibody (mAb) against PC, suggesting that they are glycoproteins. Exposure of PC-ES to N-glycosidase F results in weak and inconsistent loss of binding of the mAb, indicating that unlike the situation with respect to ES of Acanthocheilonema viteae, PC is highly unlikely to be solely attached to N-type glycans. Conversely, treatment of O. gibsoni PC-ES with mild alkali, a strategy for removing O-type glycans, abolishes mAb binding. These results suggest that PC may be attached to O. gibsoni proteins mainly via O-type glycans, and raise the possibility that filarial parasites may vary with respect to their mode of attachment of PC. The implications of this with respect to the design of inhibitors of PC attachment for use as anti-filarial drugs, are discussed.

Amidohydrolases↗
PubMed1979

The development of Onchocerca ochengi (nematoda: filariodea) to the infective stage in Simulium damnosum s.l. with a note on the histochemical staining of the parasite.

Onchocerca ochengi, an intradermal filarial parasite of cattle in Africa, was shown to develop normally to the infective stage in a member of the Simulium damnosum complex, almost certainly S. sanctipauli. The development of the parasite in the thoracic musculature and head of the fly was completed in 6 days at an ambient temperature of 25-30 degrees C. The larvae were very similar in rates of development, morphology and distribution to those of the human parasite O. volvulus in the same vector. No histochemical differences were found in the enzyme staining of O. ochengi distinct from the pattern described earlier for O. volvulus.

Animals↗
PubMed1997

Ultrastructure of infective larvae (L3) of Onchocerca volvulus (Nematoda: Filarioidea) developed in Simulium yahense in Liberia.

Third-stage infective larvae of Onchocerca volvulus were examined to elucidate the ultrastructure and the interrelations of the stoma, esophagus, intestine, and nervous system. The alimentary canal involves a cuticularized stoma with a triradiate lumen that is continuous with a similar triradiate lumen in the muscular region of the esophagus. The lumen wall may be laterally appressed or opened into a stellate form in the glandular region. Posteriad from the esophagointestinal valve, the cylindroid lumen becomes partially occluded with microvilli formed by the evaginations of the apical membranes of the intestinal epithelium. Cross sections, through this region reveal that groups of 5 radiating epithelial cells are joined near the lumen surface by junctional complexes. The alimentary canal terminates via a rectal valve and channel supported by somatic and neural cells. The central nervous system consists of a nerve ring that surrounds the muscular region of the esophagus. Related neurons support chemoreceptors and tactoreceptors of sensilla and the extensive coelomyarian and meromyarian somatic muscles. Extensive accumulations of glycogen rosettes are present in many of the muscle and hypodermal cells.

Animals↗
PubMed1997

Characterization of a recombinant Onchocerca volvulus antigen (Ov33) produced in yeast.

A yeast (Saccharomyces cerevisiae) expression system has been adapted to produce reagent quantities of a major Onchocerca antigen, Ov33. Using a pool of monoclonal antibodies produced against third-stage larvae, a cDNA library constructed from adult O. volvulus worms was screened. Twenty-seven cDNAs were isolated, two of which had sequence homology to Ov33, a putative aspartyl protease inhibitor, which is the immunodominant antigen of O. volvulus. These cDNAs were expressed at high levels intracellularly or through the secretory pathway of S. cerevisiae. Localization studies using antisera produced against purified recombinant protein demonstrated that Ov33 is a very abundant parasite protein present in the hypodermis, muscle, and uterus of female worms, as well as in embryonic microfilariae. The soluble recombinant protein secreted by yeast (C71) demonstrated inhibitory activity against the aspartyl protease pepsin. Antibodies to the recombinant protein-mediated leukocyte adherence to and killing of skin microfilariae. The sensitivity of a diagnostic test using recombinant Ov33 was evaluated using sera from 441 patients. The mean sensitivities for the two recombinant constructs, C27 and C71, were 82.2% and 85.4%, respectively. The combined sensitivity using both recombinant proteins was 94%.

Animals↗