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PubMed1991

Eosinophil-larval-interaction in onchocerciasis: heterogeneity of in vitro adherence of eosinophils to infective third and fourth stage larvae and microfilariae of Onchocerca volvulus.

Adherence of eosinophilic granulocytes from patients with onchocerciasis to microfilariae (Mf), third (L3) and fourth (L4) stage larvae of Onchocerca volvulus was studied in vitro. Native and heat-inactivated sera from patients with onchocerciasis (OS), from endemic controls without signs of the disease (ECS), from healthy Caucasians (NS) or foetal calf serum (FCS) served as sources for adherence mediating factors. In FCS-supplemented medium eosinophils did not adhere to any larvae. None of the sera mediated the adherence of eosinophils to L4. Eosinophils adhered to L3 in the presence of OS, ECS and NS, whereas OS exclusively mediated adherence to Mf. Reduced adherence rates of eosinophils to L3 occurred in heat-inactivated or zymosan-activated OS, ECS or CS. Eosinophils bound to the L3 cuticle of moulting stage but not to the newly exposed L4 cuticle. A single adherent layer of effector cells was found around cast L3 cuticle, multiple layers were found around intact L3 leading to subsequent paralysis of the larvae and to an amplification of the toxic effector potential by homotypic intereosinophilic adhesion. Our experiments document heterogeneity of in vitro effector cell adherence to the three larval stages of O. volvulus and indicate that complement-dependent as well as independent mechanisms are operative in eosinophil-larval-interaction. The results emphasize the importance of the invading infective larval stages of O. volvulus as possible targets for vaccine production.

Animals↗
PubMed1988

Investigations of enzymatically isolated male Onchocerca volvulus: qualitative and quantitative aspects of spermatogenesis.

A simple technique for the identification of the different spermatogenetic stages of Onchocerca volvulus is described. It is practicable for large-scale examinations in the field. The reproductivity of male worm populations can be assessed and directly correlated with the corresponding female worm populations. The activity of male filariae decreases with the increasing age of the worm population. Only few male worms transmit spermatozoa during mating, but spermatogenesis is continous also in very old male individuals. These old male filariae store high numbers of spermatozoa and cease their migration between the nodules.

Aging↗
PubMed1998

Onchocerca volvulus: ultrastructural localization of two glutathione S-transferases.

Glutathione S-transferases (GSTs) are essential detoxification enzymes for virtually all cells and may additionally aid in parasite survival by counteracting host-induced damage. GSTs from parasitic nematodes have been identified as potential targets for both immuno- and chemotherapy. To more closely characterize a 31-kDa (OvGST1) and a 24.5-kDa (OvGST2) GST from the pathogenic human filarial parasite Onchocerca volvulus, immunolocalization by electron microscopy was performed using two distinct affinity-purified polyclonal antisera raised against the recombinant OvGST1 and OvGST2. The strongest immunogold staining for OvGST1 was identified in the body wall of adult worms, especially in protuberances of the cuticle which lie in pouches of the hypodermis and in the outer zone of the syncytial hypodermis, where the external plasma membrane forms series of lamellae. Gold particles were also observed on the epicuticle of the adults and in the region of the border between the cuticle and hypodermis of microfilariae. The larval stages L1, L2, and infective L3 were also immunopositive for OvGST1. There was no specific labeling in the longitudinal musculature, the intestine, or the uterine wall of the adult worm. In contrast to the results for OvGST1, immunogold labeling for OvGST2 was observed throughout the whole hypodermal cytoplasm. The epithelial cells of the uterine wall showed moderate labeling. These ultrastructural immunolocalization results are consistent with the molecular characterization of both enzymes, indicating that OvGST1 is secreted out of the hypodermis into the cuticle and is acting at the host-parasite interface, while OvGST2 functions as an intracellular cytosolic housekeeping enzyme.

Animals↗
PubMed1983

Onchocerca volvulus: detection of circulating antigen by monoclonal antibodies in human onchocerciasis.

A monoclonal antibody of the IgM class recognizing Onchocerca volvulus circulating antigen (COA) was obtained. This monoclonal antibody was used in a radioimmunoprecipitation-PEG assay (RIPEGA) to detect circulating antigen in onchocerciasis patients' sera. COA could be detected in 63 (80%) of the 79 African patient sera tested, and in 126 (76%) of the 164 Indian (Venezuela) sera studied. There was no direct correlation between the presence of COA detected in the patient serum and the level of microfilarodermia. The RIPEGA using this monoclonal antibody detected COA in 91% of children under 10 years old, whereas the microfilarodermia in this group was positive in only 52% of the cases. The specificity of this test is improved compared to the results obtained with polyclonal antibodies. Immunofluorescence studies suggest that the COA might be located in the microfilaria cuticle.

Adolescent↗
PubMed1994

[Onchocerciasis control program in West Africa: socioeconomic development and risk of recrudescence of transmission. 2. Experimental study of the transmission of Onchocerca volvulus strains from Southwestern Sierra Leone by Simulium yahense and Simulium squamosum].

The movements of human populations towards the mining wealth of the northern parts of Sierra Leone are favorable to a high contact rate between onchocerciasis patients coming from the south-western area of this country and the vector species Simulium yahense and Simulium squamosum which assume the essential of onchocerciasis transmission in the above-mentioned mining area. In fact, the Onchocerca volvulus strains concerned by this contact seem to be more pathogenic than those locally transmitted. In order to assess the danger it could represent for the Onchocerciasis Control Programme in West Africa, we carried out the experimental study of transmission which may result from this contact when more or less infected onchocerciasis patients are involved. The results indicated that this transmission by S. yahense may reach high proportions only when heavily infected onchocerciasis patients are implicated. We took also notice of the low capacity of S. squamosum to transmit the O. volvulus strains from the south-western Sierra Leone, irrespective of the microfilarial load of patients. Thus, in the most favorable conditions of a high parasite-vector contact of the study, involvement of S. yahense and onchocerciasis patients with high skin microfilarial loads is the only occurrence to which a high risk of intensive transmission may be related. The authors consider that the probability of such a risk occurring will be drastically reduced, due to the considerable decrease of skin microfilarial loads in human communities which regularly have the advantage of ivermectin (Mectizan) mass treatments.

Animals↗
PubMed1991

cDNA clones of Onchocerca volvulus low molecular weight antigens provide immunologically specific diagnostic probes.

We report here a panel of cDNA clones from Onchocerca volvulus which were isolated on the basis of being uniquely recognised by onchocerciasis sera and not by sera from patients infected with the major lymphatic filarial nematode parasite Wuchereria bancrofti. Over 90% of O. volvulus recombinants from a primary screen were found to cross-react with lymphatic filariasis sera and were discarded. The subset of specific clones, selected with pooled sera, was then screened with panels of individual patient sera. Individual onchocerciasis cases showed a highly heterogeneous pattern of recognition of recombinant peptides, but several clones were identified which could be combined in a cocktail of antigenic epitopes to successfully detect all infected cases in the study. All these clones encode low molecular weight proteins of the parasite, confirming earlier reports that antigens of this size class show greater species specificity. Several clones encode proteins of 20-23 kDa, the same molecular weight range as the major surface protein of adult worms. The two most commonly recognised clones, Ov22/31M and Ov20/36M were subcloned into the vector pNGS 8 which produces fusion proteins attached to a polyasparagine leader. The fusion peptides of both Ov22/31M and Ov20/36M were soluble and easily purified by gel filtration. Purified fusion protein was used in ELISA to assess reactivity of infected patients giving 90% sensitivity with 100% specificity.

Animals↗
PubMed1992

Analysis of antibody responses directed against two Onchocerca volvulus antigens defined by monoclonal antibodies.

Monoclonal antibodies, reactive with antigens solubilised from the body wall of intact female Onchocerca volvulus using 2% 2-beta-mercaptoethanol, have been characterised. Two IgG1 antibodies, Cam1 and Cam28, recognised antigens of apparent molecular weights of 18,000 and 28,000; and 120,000, respectively. The target antigens of Cam1 and Cam28 could be localised in the cuticle. Inhibition ELISAs showed that target epitopes of both monoclonal antibodies induce an antibody response in onchocerciasis patients. 153 sera from Sierra Leonean patients were tested for their individual antibody levels against antigen epitopes recognised by Cam1 and Cam28. Patients within the age of 5-8 years had the highest levels of antibodies against the Cam28-epitope, whereas patients above 60 years had almost none. Amicrofilaremic patients had higher anti-Cam28 antibody levels than microfilaremic patients and there was a significant difference between groups with no chronic skin disease and those with mild or severe signs. A high percentage of patients (80.4%) recognised the Cam1-epitope, highest antibody levels being found in patients within the age group of 15 to 45 years and in microfilaremic patients. However, levels of antibodies inhibiting monoclonal antibody Cam1 could not be correlated with presence or absence of skin disease.

Adolescent↗
PubMed1984

Homologous and distinctive antigens of Onchocerca volvulus and Dirofilaria immitis: detection by an enzyme-linked immuno-inhibition assay.

The limited accessibility of specimens of Onchocerca volvulus has hampered investigations of factors that determine the clinical course of infection of man by this filarial nematode. Parasites antigenically related to O. volvulus have been variously proposed as alternative sources of test antigens. The present study has utilized serologic methods to probe the degree of identity of the antigenic constituents of O. volvulus and Dirofilaria immitis, the dog heartworm. 35S-methionine-labeled proteins of dog heartworm microfilariae (mf) were analyzed in immunoprecipitation assays by using a panel of sera drawn from humans diagnosed for onchocerciasis and from canines infected with D. immitis. Unique and common antigens detected by these sera were resolved in polyacrylamide gels. The majority of high-titered human and infected dog sera reacted with antigenic molecules estimated at 15, 16, 28, 42, 54, 66, and 100 kilodaltons. In addition, a subset of these sera detected large amounts of a 75 kilodalton antigen. Human anti-O. volvulus antibodies were found to bind to intact glutaraldehyde-fixed mf of D. immitis in immunoperoxidase assays. In enzyme-linked immunosorbent assays, the same sera reacted with extracts of three distinct developmental stages of the canine pathogen--namely, the mf, embryonated eggs, and adult worms. Preincubation of dirofilarial antigens with human anti-O. volvulus sera inhibited the binding of dog anti-heartworm serum by as much as 70%. Sera of guinea pigs hyperimmunized with mf of O. volvulus inhibited the binding of the immune dog serum to the same extent. These results represent evidence for extensive homology in the antigenic composition of O. volvulus and D. immitis.

Animals↗
PubMed1989

A preliminary assessment of the feasibility of evaluating promising antifilarials in vitro against adult Onchocerca volvulus.

The suitability of motility indices and tetrazolium-based colorimetric assays for the determination of the viability of adult Onchocerca volvulus after in vitro exposure to potential macrofilaricides has been examined. Experimentation showed that both techniques could be applied to adult O. volvulus, although the variability between individual worms necessitated the use of large experimental groups. The potential of using cut anterior tips of female O. volvulus for screening was also investigated. These were shown to give reasonably consistent motility indices, and drug effects were discernible even after 72 h in vitro culture. Application of these viability criteria to studies on the short-term in vitro survival of intact male and female O. volvulus incubated in Eagles MEM plus serum, under 5% CO2 in air, showed this medium to be suboptimal with a greater than 50% loss of worm viability within 144 h of nodulectomy. Males isolated by the collagenase technique were shown to be significantly less viable than dissected males, by both motility indices and tetrazolium reduction. The results highlight the need to use either dissected males, or in the case of females, the need to minimize exposure to collagenase solution. A possible mechanism for selecting a more uniformly viable female worm population is discussed. Examination of the in vitro effects of CGP 20376 using these viability criteria/assay systems showed some delayed suppression of worm motility, but after 120 h in vitro CGP 20376 was not macrofilaricidal against male or female O. volvulus. Male worms were also implanted subcutaneously into gerbils.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Characterization of genes encoding members of the nuclear hormone receptor superfamily from Onchocerca volvulus.

Several lines of evidence suggest that molting in parasitic nematodes is controlled through the action of steroid molting hormones, or ecdysones. In other organisms, the central mediator of steroid hormone action is the hormone receptor. These receptor molecules are members of a superfamily of proteins called the nuclear hormone receptor family. Using an oligonucleotide derived from the amino-acid sequence of the Drosophila melanogaster ecdysone receptor, genes encoding homologues of the nuclear hormone receptor family were identified in the genome of the human filarial parasite Onchocerca volvulus. The O. volvulus genome contains at least three genes that encode putative members of the nuclear hormone receptor superfamily. A complete cDNA for one of these genes, designated OvNHR-1, has been isolated and characterized. The OvNHR-1 cDNA was 2378 bp in length, and contained a single open reading frame of 1104 bp. The open reading frame encoded a peptide with all of the features characteristic of a member of the nuclear hormone receptor superfamily of proteins. OVNHR-1 appeared to be encoded by a single-copy gene. Expression of the mRNA corresponding to OvNHR-1 was developmentally regulated, with maximal expression occurring during early embryogenesis. The polypeptide encoded by the OvNHR-1 open reading frame is antigenic in a minority of individuals exposed to O. volvulus.

Amino Acid Sequence↗
PubMed1993

Onchocerca volvulus provides ligands for the stimulation of human gamma/delta T lymphocytes expressing V delta 1 chains.

Peripheral blood mononuclear cells (PBMC) from 8 onchocerciasis patients, treated or not with ivermectin, were analyzed for phenotypic cell surface markers. A significant increase (P < .05) in gamma/delta T cells expressing the V delta 1 chain compared with normal and endemic controls was detected in all patients. PBMC populations from onchocerciasis patients were not expanded after restimulation with Onchocerca volvulus antigens in vitro, but both V delta 1 and V delta 2 T cells from normal donors were increased significantly in response to O. volvulus and Mycobacterium tuberculosis (P < .05), respectively. Frozen sections of all 5 onchocerca nodules tested demonstrated an increased number of CD3+ cells in the vicinity of the adult worm, in all cases expressing the alpha/beta T cell receptor and in 2 patients also expressing the gamma/delta T cell receptor; 60% of T cells expressed the activation marker Ki67. These data suggest that O. volvulus provides ligands to V delta 1 T cells.

Adult↗
PubMed1990

The transmission of onchocerciasis at a forest village in Sierra Leone. I. Simulium damnosum s.l. biting densities and infection with Onchocerca volvulus at five representative sites.

Previous studies in the forest area of Sierra Leone have shown that transmission of Onchocerca volvulus takes place many kilometers away from large breeding rivers and sometimes in open farmland. To determine where and when people in a forest village were most likely to be infected, catches of Simulium damnosum s.l. were carried out every week for 12 months, at five sites in and near a village where onchocerciasis was mesoendemic. The number of flies caught per man a day at open farm sites was significantly higher than the number caught at riverside sites. Infective flies were caught only in farmland and only during the early rainy season. The combined Annual Transmission Potential for the five sites was 129 larvae per man per year. Isoenzyme electrophoresis and morphology of biting flies identified the S. sanctipauli/soubrense subcomplex as the most common vector species. Simulium yahense and S. squamosum were sometimes present. It was concluded that the classical riverside monitoring sites do not represent high risk areas for the transmission of onchocerciasis in a forest village sited well away from the main S. damnosum s.l. breeding sites. The highest risk areas are in open farmland.

Animals↗
PubMed1995

Molecular cloning of a gene expressed during early embryonic development in Onchocerca volvulus.

Little attention has been paid to the reproductive biology of filarial nematode parasites as a possible target for immunological or chemotherapeutic intervention. An interruption of the reproductive process would, in addition to breaking the cycle of transmission, reduce the morbidity associated with certain filarial infections. As part of our efforts to define molecules that have important functions during filarial embryogenesis, antibodies against embryo-associated proteins were used to identify a 6308-bp cDNA sequence (ovt1) from an Onchocerca volvulus cDNA expression library. The ovt1 cDNA contained an open reading frame that coded for 2022 amino acids. The deduced amino acid sequence was highly hydrophilic, alpha-helical in nature and included two leucine zipper domains. OVT1 also contained a single Arg-Gly-Asp (RGD) site. The results of Southern blot analyses demonstrated that an ovt1-like gene occurs in a number of different species of filarial nematodes. In situ hybridization experiments to identify tissues that contain ovt1 transcripts showed that ovt1 was transcribed at high levels in the late morula/early blastocyst stage of embryonic development. Transcripts for ovt1 were also detected in O. volvulus larvae and in the hypodermal cells of adult parasites. Two fragments of ovt1 were expressed as fusion proteins and the fusion proteins were used to produce antibodies in rabbits. Both antibodies recognized a native protein with an apparent molecular mass of 230 kDa in extracts from gravid female O. volvulus. In addition, the antibodies reacted with a restricted number of lower-molecular mass bands which may represent the products of post-transcriptional or post-translational processing. The predicted coiled-coil structure and the sites of transcription suggest that OVT1 may be a component of the extracellular matrix.

Amino Acid Sequence↗
PubMed1994

The Onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase.

Protein disulfide isomerase (PDI) functions to catalyze the formation of correct disulfide bonds in nascent proteins, and also acts as one of the subunits of prolyl-4 hydroxylase, the enzyme responsible for the oxidative maturation of procollagen. Since the cuticle of parasitic nematodes consists primarily of a network of collagen molecules which are connected through intermolecular disulfide bonds, PDI might be expected to be involved in the process of cuticle biosynthesis. The isolation and characterization of a cDNA encoding the PDI homologue of Onchocerca volvulus is described. This cDNA contains a single, long open reading frame that encodes sequence motifs identical to the two known active sites of PDI for isomerase activity. The O. volvulus PDI appears to be encoded by a single copy gene. Both in situ hybridization and immunolocalization data suggest that PDI is both spatially and temporally regulated in O. volvulus. The pattern of spatial and temporal regulation is consistent with the involvement of PDI in the biosynthesis of the parasite cuticle. The parasite protein appears to be an antigen recognized by a minority of individuals exposed to O. volvulus.

Amino Acid Sequence↗
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↗
PubMed2003

Entomological evaluation by pool screen polymerase chain reaction of Onchocerca volvulus transmission in Ecuador following mass Mectizan distribution.

The prevalence of infected and infective black flies was estimated by pool screen polymerase chain reaction (PCR) amplification in the three river basins in Ecuador endemic for onchocerciasis. Mass distribution of ivermectin (Mectizan) resulted in dramatic declines in the prevalence of infected and infective flies. In the Rio Santiago river basin, no infections were detected, suggesting that transmission had ceased. The ratio of infected to infective flies in Simulium exiguum was 10-fold lower than the corresponding ratio for Simulium quadrivittatum, suggesting that S. exiguum is a more-competent vector for Onchocerca volvulus than S. quadrivittatum. However, the prevalence of infective flies in the two species was not different, suggesting that S. quadrivittatum may play an important vectorial role where it is the dominant human-biting species. The data demonstrate that pool screen PCR is an efficient way to monitor transmission in areas subject to control, and to certify an area as free of O. volvulus transmission.

Animals↗
PubMed1992

Experimental onchocerciasis in chimpanzees. Antibody response and antigen recognition after primary infection with Onchocerca volvulus.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.

Animals↗