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PubMed1980

Simulium exiguum, the vector of Onchocerca volvulus on the Rio Micay, Colombia.

Simulium exiguum were collected in the San Antonio area on the Rio Micay as they blood-fed on a volunteer naturally infected with Onchocerca volvulus. The daily dissection of specimens revealed that delayed larval development was common and 23% of the larvae were abnormally formed. However, third-stage larvae were recovered by late day 5 (140 hours) following engorgement. It is concluded that S. exiguum is the vector of O. volvulus at this Colombian endemic focus even though its host efficiency is somewhat limited. S. metallicum collected near Cali from a naturally infected San Antonio volunteer was shown to be a relatively poor experimental host for O. volvulus.

Aged↗

PubMed1988

Parasite antigens are present in breast milk of women infected with Onchocerca volvulus.

We used a noncompetitive two-site ELISA with 5 monoclonal antibodies to determine whether parasite antigens are present in breast milk from women infected with Onchocerca volvulus. Seven out of 13 available milk samples contained significant amounts of filarial antigens. Antigen indices in milk correlated with levels of microfilarodermia (Rs = 0.74, P less than 0.005). Antigen-containing milk samples markedly inhibited mitogen-induced proliferation of human mononuclear cells and activated cells within this population that suppressed the proliferative response of autologous lymphocytes to mitogens and antigens. These findings indicate that parasite products are present in breast milk of O. volvulus-infected women and suggest that these may induce immune tolerance and/or suppression in infants born of infected mothers.

Animals↗

PubMed1990

Identification of an Onchocerca volvulus cDNA encoding a low-molecular-weight antigen uniquely recognized by onchocerciasis patient sera.

The primary structure of an immunodominant antigen of the filarial parasite, Onchocerca volvulus was deduced from cDNA sequence analysis. Using affinity-purified antibody from onchocerciasis patients from West Africa, we have isolated a cDNA clone from a lambda gt11 cDNA expression library derived from microfilariae-producing female O. volvulus. The open reading frame encodes 152 amino acids, and the deduced sequence predicts a Mr of 16,850 (consistent with the apparent Mr of 18,000 of the immunoprecipitated in vitro translated product). The primary translation product contains a putative signal peptide of 16 amino acids. The mRNA coding for this antigen has an estimated size of 950 nucleotides. Furthermore, immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis, the cuticle, and in the uterus of the filarial worms. Since this antigen is recognized exclusively by sera from onchocerciasis patients, and not by other sera from patients infected by other filarial parasites, it may prove to be an especially valuable tool for improving the specific diagnosis of onchocerciasis.

Amino Acid Sequence↗

PubMed1990

Onchocerca volvulus: biochemical and morphological characteristics of the surface of third- and fourth-stage larvae.

The annulated cuticles of third- and fourth-stage larvae of Onchocerca volvulus have the typical structure of other nematodes but the cuticle of fourth-stage larvae was thinner. The surface of the third-stage larva was wrinkled and fuzzy, while that of the fourth-stage was smooth. Intermediate stages in the formation of the new cuticle and epicuticle beneath the old basal layer and of the separation of the cuticles are shown. Monoclonal antibodies specific to the surface of third-stage larvae did not react with the surface of the fourth-stage larvae. Binding of the monoclonal antibodies to the third-stage larvae was abrogated by treatment of the worms with trypsin and proteinase K, but was unaffected by treatment with periodate or the detergents sodium deoxycholate and SDS. The lectins RCA120 and WGA, but not any of the other lectins tested, bound only to the surface of fourth-stage larvae, and not to that of third-stage larvae. The surfaces of third- and fourth-stage larvae were shown to be different and contained stage-specific surface epitopes.

Animals↗

PubMed1991

Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms.

The isolation and characterization of a recombinant cDNA clone (OV7) expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Using chimpanzee antiserum generated against irradiated infective larvae, we isolated a cDNA clone from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA. The open reading frame encodes 131 amino acids corresponding to a 15.2-kDa protein. Affinity purified antibodies which bound specifically to OV7 fusion polypeptide recognized a single antigen with an apparent molecular weight of 17,000 in extracts of L3, L4 and adult worms. Immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis and the basal layer of the cuticle of L3 and female adult worm, and in the egg shell around developing microfilariae. Since the OV7 fusion polypeptide is onchocerca-specific and is recognized specifically by sera from onchocerciasis patients, and sera from non-patent but infected chimpanzees, and not by sera from patients with other filarial parasites, it may have potential as an antigenic component in a test for detection of non-patent and patent infections of O. volvulus. The OV7 amino acid sequence contains residues that have a probable homology with the cysteine proteinase inhibitor superfamily.

Amino Acid Sequence↗

PubMed1984

Descriptive statistics for the larval stages of Onchocerca volvulus in host-seeking Simulium ochraceum.

In four localities of Guatemala with dissimilar intensities of onchocerciasis, filarial larvae were recovered by dissection from host-seeking Simulium ochraceum. Measurements made on over 600 Onchocerca volvulus larvae were used to characterize the early first, late first, second, and third developmental stages. The numerical attributes used to characterize the third larval stage and the associated means were body length (657.3 microns); anterior body width (18.9 microns); posterior body width (20.2 microns); distance from anterior end to nerve ring (87.9 microns), to junction of muscular and glandular esophagus (137.6 microns), and to junction of esophagus and intestine (419.6 microns); and distance from anus to tip of tail (36.8 microns). Ratios to total body length were as follows: distance from anterior end to nerve ring, mean = 0.133, to junction of esophagus and intestine, mean = 0.634, and to anus, mean = 0.945. Differences between these phenotypic features and those reported for African O. volvulus appeared to be insufficient to distinguish the two forms. With very few exceptions, the filarial larvae found in host-seeking S. ochraceum were considered to be O. volvulus.

Anal Canal↗

PubMed1991

Immunity to onchocerciasis: recognition of larval antigens by humans putatively immune to Onchocerca volvulus infection.

Immunoblot analyses were done using sera from 12 individuals without evidence of onchocerciasis and 16 with active infection from an area of Guatemala holoendemic for onchocerciasis. For adult antigens from Onchocerca volvulus, no differences in antigen recognition could be identified between the two groups. In contrast, when infective larval (L3) antigen preparations derived from the related animal parasite Onchocerca lienalis were used, IgG from the "immune" individuals preferentially recognized a 45- to 50-kDa triplet and a 22-kDa L3 antigen. When L3 antigens of Brugia malayi were used, sera from putatively immune individuals identified a high-molecular-weight triplet/quadruplet plus several additional antigens of lower molecular weights that were recognized by sera from few (or none) of the infected patients. These findings define some differences in antibody specificity in onchocerciasis patients and therefore might define potential target antigens of humoral host defense. The exact nature of such defenses is unknown.

Adolescent↗

PubMed1980

Analysis of enzymatically isolated adults of Onchocerca volvulus after treatment of patients with suramin or metrifonate.

The application of a new technique is described permitting a better recognition of activities of drugs on macrofilariae of Onchocerca volvulus. In two villages of a hyperendemic focus in Liberia onchocercomata were excised from untreated persons and from patients treated with conventional doses of suramin or metrifonate. The adult worms were enzymatically isolated with collagenase and microscopically examined for degenerative alterations including calcifications and for the presence of eggs and embryos in the female parasites. Six weeks after the last dose of suramin the degenerative alterations could not be distinguished qualitatively from those in untreated worms, but a statistically significant macrofilaricidal efficacy could be shown by quantitative analyses, although more than half of the parasites were still alive at this early date. Metrifonate did not show any macrofilaricidal effect.

Animals↗

PubMed1989

Evaluation of a specific enzyme immunoassay for onchocerciasis using a low molecular weight antigen fraction of Onchocerca volvulus.

A micro-ELISA technique has been developed for the diagnosis of onchocerciasis. With the use of a low molecular weight (LMW) fraction of a soluble, adult Onchocerca volvulus extract as an antigen, the problem of nonspecificity observed with the crude worm extract is negligible. Seventy-three sera from proven cases of other filarial infections were weakly reactive or nonreactive. From cross-sectional and longitudinal surveys in a West African village hyperendemic for onchocerciasis, we concluded that this assay detects antibodies elicited when female worms start to produce microfilariae. Thirty-two of 33 children aged less than or equal to 15 years with positive skin snips were seropositive. High antibody levels could also be found in children who only became parasitologically positive 1-4 years after seroconversion. This leads to the conclusion that serology is more sensitive than the 2-snip method. In individuals with chronic infection (exposed for greater than 15 years), antibody titers were consistently lower (independent of the density of skin microfilariae) than in the early phase of a patent infection. A comparative serological study in a mesoendemic village revealed that the assay can serve to assess onchocerciasis prevalence by testing individuals aged 5-15 years.

Adolescent↗

PubMed1981

The effect of drug treatment on the pattern of emergence of Onchocerca volvulus microfilariae from skin snips.

A controlled study was carried out to determine the effect of some known anti-filarial drugs on the pattern of emergence of Onchocerca volvulus microfilariae from skin snips into saline. The results show that before treatment, 81.7 to 92.1% of microfilariae emerge in all the groups within 24 hours. The differences between the groups were not significant. After treatment with oral diethylcarbamazine, metrifonate and topical diethylcarbamazine (DEC) the percentage of microfilariae emerging from the snips dropped significantly to 48.4, 56.0 and 60.1 respectively, whereas no significant change occurred following treatment with levamisole (88.1%) and yeast tablets (89.1%). A pair-wise comparison of the post-treatment microfilarial emergence in all the groups showed statistically significant differences when (i) oral DEC is compared to levamisole, topical DEC and yeast, (ii) metrifonate is compared to levamisole and yeast and (iii) topical DEC is compared to yeast. It is suggested that microfilariae which fail to emerge from snips after treatment are those that have been killed or paralysed by the action of the drugs. The study has thus established that drug action in vivo is one of the factors which affects the proportion of microfilariae which emerge from skin snips and provides an additional parameter for measuring the antimicrofilarial potential of drugs in clinical chemotherapeutic trials.

Animals↗

PubMed1992

Identification and characterization of an Onchocerca volvulus cDNA clone encoding a microfilarial surface-associated antigen.

The identification and characterization of a recombinant cDNA clone (OV103) expressing a microfilarial surface-associated antigen of Onchocerca volvulus is described. OV103 was identified and isolated from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA using a chimpanzee antiserum, taken 2 years after infection with third-stage larvae of O. volvulus. The cDNA clone encodes a 12.5-kDa protein that corresponds to a 15-kDa parasite protein present in microfilariae and adult female worms. The antigen encoded by this clone is located in the basal layer of the cuticle and the hypodermis of the female adult worm, and on the surface of microfilariae. OV103 fusion polypeptide is recognized only by some sera from onchocerciasis infected subjects (57%), but more significantly (89%) by sera from individuals that have low levels of patent infection. In addition, the antibody response to this protein developed before appearance of microfilariae in the skin of chimpanzees that had developed non-patent or low level patent infections, while the antibody response in chimpanzees with high levels of microfilariae appeared later at the time of appearance of microfilariae. Preliminary experiments indicated that affinity purified antibodies directed against OV103 fusion polypeptide mediated killing of nodular microfilariae in vitro in the presence of normal peripheral blood granulocytes.

Amino Acid Sequence↗

PubMed1989

Studies on the activity of the Ciba Geigy compounds CGP 6140, 20376, 20309 and 21833 against third and fourth stage larvae of Onchocerca volvulus.

The effect of four new antifilarial compounds, (CGP 6140, 20376, 20309 and 21833, Ciba Geigy Ltd. Basle) on the viability of third and fourth stage larvae of Onchocerca volvulus was tested in vitro and in vivo. The motility of the larval stages and the moulting process from the third to the fourth stage were used as parameters for the drug activity in vitro. All four compounds showed good effects in vitro on the moulting rates compared to the controls after incubation at different concentrations in culture medium with serum. After an exposure for three hours to different concentrations of the compounds in medium without serum, only CGP 20376 had a clear inhibitory effect on the developing larvae. The moulting rate was reduced by this compound to 20% of the control values with 1 microgram/ml, a concentration that is comparable to plasma levels reported from chimpanzees. In both experimental situations, with serum and without serum, the motility of the developing larvae was only affected at very high concentrations of the compounds. The results obtained with fourth stage larvae were comparable. CGP 6140 and CGP 20376 were tested in vivo, using a diffusion chamber technique for the implantation of infective larvae into the peritoneum of Mastomys natalensis and assessing the survival of the implanted parasites. High doses of CGP 6140 had only unsatisfactory effects on the larvae in this model system whereas CGP 20376 had inhibited moulting and killed most larvae with a daily dose of 25 mg/kg for five consecutive days.

Animals↗

PubMed1991

Onchocerca volvulus: characterization of monoclonal antibodies reactive with surface components of third-stage larvae.

Five murine monoclonal antibodies (Mab's) have been produced which are reactive with the surface of the third-stage larvae (L3) of Onchocerca volvulus. These were produced from a fusion performed after intrasplenic injection of 10 live O. volvulus L3. Hybridomas were first screened by Elisa using a PBS extract of O. volvulus female adult worms. Elisa negative wells were screened by IFA on whole formalin-fixed L3. Five Mab's were isolated which were reactive with the surface of L3, all were found to be of the IgM isotype. All five Mab's were cross-reactive with the surface of O. lienalis L3, but not with the L3 of Brugia malayi, B. pahangi, Dirofilaria immitis, or Acanthocheilonema viteae. One of the Mab's, OV3-9, reacted with an antigen common to many species of helminths. The other four were genus specific reacting only with O. volvulus and O. lienalis. All 5 Mab's reacted with L2 of O. volvulus, but not with the L4 by IFA on whole fixed larvae. Only Mab's OV3-8 and OV3-9 reacted with cryosections of adult O. volvulus by IFA indicating that the other 3 Mab's are specific for the surface of L2/L3 of the genus Onchocerca. None of the Mab's reacted with detergent dissociated L3 proteins in Western Blot analysis. The ability of L3 to bind the Mab's was abolished by incubating larvae in either proteinase K or trypsin but was unaffected by incubation of larvae in detergents, or by treatment with periodate, indicating the proteinaceous nature of the Mab specific epitopes. Anti-phosphorylcholine Mab (Gib-13) did not react with the surface of L3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1968

The effects of drugs on Onchocerca volvulus. 1. Methods of assessment, population dynamics of the parasite and the effects of diethylcarbamazine.

It is important to standardize quantitative methods for assessing the action of drugs on Onchocerca volvulus in man. There are certain disadvantages to making observations on adult worms removed from excised nodules in treated patients, but a great deal of information on drug action can be obtained by making a careful study of the concentrations of microfilariae in multiple weighed skin snips taken before treatment and at intervals after treatment.Before drug trials can be carried out intelligently by this method it is necessary to know the normal length of life of the various stages of the parasite in man. By a variety of experimental methods the life-spans of the adult worms and the microfilariae have been determined, as well as the duration of the prepatent interval.Diethylcarbamazine can be used at doses that are effectively microfilaricidal but have no action on the adult worms. This drug can therefore be used to eliminate any residual microfilariae that remain after treatment with a new drug under trial, thus permitting a more rapid assessment of the latter's action on adult worms.

Adolescent↗

PubMed1975

The fate and migration of microfilariae of a Guatemalan strain of Onchocerca volvulus in Simulium ochraceum and S. metallicum, and the role of the buccopharyngeal armature in the destruction of microfilariae.

The migration and fate of microfilariae of a Guatemalan strain of Onchocerca volvulus in Simulium ochraceum and S. metallicum was studied. The anatomical distribution of microfilariae at various times after ingestion was recorded from a study of histological sections of the flies. S. ochraceum showed higher microfilarial intakes than S. metallicum which is probably attributed to an inherent difference in their ability to ingest. The migration of microfilariae out of the mid-gut took place essentially through the expanded part (the stomach). Despite the high microfilarial intakes by S. ochraceum, only a few microfilariae were able to migrate successfully into the thorax (2.6%) whereas in S. metallicum showing a low microfilarial intake, a higher rate of migration was noted (74.5%). The main bulk of microfilariae ingested by S. ochraceum died and disintegrated in the stomach. This was due to the presence of a well-developed cibarial armature in the buccopharyngeal apparatus of S. ochraceum in contrast to S. metallicum where such an armature is lacking. Microfilariae were severely damaged by the cibarial teeth while en route to the stomach. They rapidly underwent profound changes in morphology and staining properties and were often surrounded by clusters of the phagocytic leucocytes in the blood mass. In some flies, microfilariae, apparently damaged, were still entangled in the cibarial teeth 5 minutes to 3 hours after taking a blood-meal.

Animals↗

PubMed1984

Deposition of eosinophil granule major basic protein onto microfilariae of Onchocerca volvulus in the skin of patients treated with diethylcarbamazine.

We investigated the association between eosinophil degranulation, as evidenced by the deposition of granule major basic protein (MBP), and the killing of microfilariae of Onchocerca volvulus in vivo following treatment with diethylcarbamazine (DEC). Utilizing an immunofluorescence procedure for the cellular and extracellular localization of eosinophil MBP in formalin-fixed, paraffin-embedded tissues, we studied skin biopsies from onchocerciasis patients before and during treatment with topically or orally administered DEC. Before DEC, there was little or no inflammatory response in either dermis or epidermis and microfilariae were essentially intact. Immunofluorescent staining for MBP revealed some filamentous fluorescence associated with dermal collagen fibers, very few eosinophils, and no fluorescence in association with intact microfilariae. In contrast, during treatment with DEC, immunofluorescent staining for MBP revealed extensive eosinophil infiltrates in both dermis and epidermis with numerous intraepidermal eosinophil abscesses containing degenerating microfilariae. An intense extracellular immunofluorescence for MBP surrounded degenerating microfilariae in the dermis and epidermis in both the presence and absence of eosinophil infiltrates as early as 4.5 hours after starting therapy. Many intact nondegenerating microfilariae were also present, but they did not show immunofluorescent staining for MBP nor a surrounding inflammatory infiltrate. The results show that immediately following administration of DEC, eosinophils localize and degranulate around microfilariae in the skin and release granule MBP onto or in close proximity to the parasite's surface. Because of the striking association between eosinophil localization, degranulation, and deposition of MBP onto microfilarial surfaces, and the degeneration of microfilariae in the skin, these observations support the hypothesis that the eosinophil, through helminthotoxic granule proteins such as MBP, damages the microfilariae of O. volvulus.

Adult↗

PubMed1987

Specific serodiagnosis with adult Onchocerca volvulus antigen in an enzyme-linked immunosorbent assay.

The cross-reactivity of the blood from onchocerciasis, loiasis, and dipetalonemiasis was tested by a micro-ELISA technique, utilizing adult Onchocerca volvulus antigen and blood samples taken on filter paper. The average ELISA values (OD at 500 nm) were as follows: 0.58 in persons with O. volvulus microfilariae (n = 81), 0.49 in microfilariae-negatives from the same endemic area (n = 39), 0.15 in dipetalonemiasis (n = 27), and 0.25 in loiasis (n = 12), while those of 65 Dipetalonema perstans-negative people were markedly low (average 0.14) and that of 22 Loa loa-negatives, 0.22, respectively. This ELISA could successfully differentiate onchocerciasis from dipetalonemiasis and loiasis.

Adolescent↗

PubMed1977

Onchocerciasis: invasion of deep organs by Onchocerca volvulus.

Autopsies of two patients with onchocerciasis in the Republic of Zaïre are reported. In one patient elephantiasis who died following diethylcarbamazine therapy there were large numbers of microfilariae of Onchocerca volvulus in the kidney, liver, pancreas, and lung. In the second patient, who also had leprosy and hyperinfection strongyloidiasis, we found an encapsulated adult O. volvulus in the wall of the thoracic aorta.

Aorta, Thoracic↗