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.     

Antigens of Onchocerca volvulus

Most studies on immunologic responses to Onchocerca volvulus have employed extracts or antigens from related filarial parasites. Consequently, little is known about the nature of O. volvulus antigens. Potential antigen sources include in vitro cultures and physicochemical fractionation of O. volvulus extracts. IgE antibody responses to a wide range of antigens occur in patients with onchocerciasis, but there is little evidence of species specificity in serologic tests. Some potent allergens are released by microfilariae, but host serum proteins appear to contaminate the most reactive fractions obtained thus far. Antibody-mediated cell adherence to microfilariae of O. volvulus occurs in vitro, and both stage and species specificity have been demonstrated. Monoclonal antibodies to O. volvulus antigens have been prepared, but, unfortunately, all to date show cross-reactivity to antigens of other filarial nematodes. Circulating antigens have been detected in patients' sera; no data are available yet on the specificity of these components. Research needs include the need for species- and stage-specific reagents for immunodiagnostic assays and for investigations on immunopathogenic mechanisms in onchocercal disease.     

Sequential affinity chromatography for the purification of antigens extracted from Onchocerca volvulus adult worms

Adult Onchocerca volvulus worms were extracted sequentially with buffers of various ionic strengths. The extract was incubated with purified human onchocerciasis immunoglobulin-G (IgG) convalently coupled to Sepharose. Antigens were then eluted with 8 M urea and 7.5 M guanidine-HCl. IgG contained in the eluates was removed by incubating the eluates with rabbit anti-human IgG covalently coupled to Sepharose. As demonstrated by double immunodiffusion and "double" crossed immunoelectrophoresis at least three antigens were isolated. Most of the antigens were totally excluded from Sephadex G-200 but entered the included volume of Sepharose 6B. In electrofocusing they focused in acid regions. Antibodies were generated in rabbits against the three antigens isolated together. The antibodies were covalently coupled to Sepharose, which was subsequently used as a new affinity medium for the isolation of antigens. Such isolated antigens appeared to be identical with those with human onchocerciasis IgG. The isolated antigens and their corresponding artificial antibodies generated in rabbits were successfully applied in the enzyme linked immunoadsorbent assay. Host material and onchocerciasis IgG left behind during the purification procedure interfered in the assay system.     

Use of recombinant Onchocerca volvulus antigens for diagnosis and surveillance of human onchocerciasis

The diagnostic value of ELISAs based on recombinant Onchocerca volvulus antigens OC 3.6 and OC 9.3 was evaluated with sera from endemic areas in West Africa, Guatemala and Ecuador. IgG assays were slightly more sensitive than those that detected IgG₄, and the antigen combination was significantly more sensitive than either antigen alone (OC 3.6, 93%; OC 9.3, 84%, combined 98%). These assays were also evaluated with sera from 2 villages in the Onchocerciasis Control Programme area of West Africa including one village (Pendie) with recent recrudescence of infection and one (Niarba) where transmission had been interrupted for 15 years by vector control. The OC 3.6 IgG antibody assay was sensitive for new infections and exposure in Pendie; 24/24 (100%) of people with positive skin snips and 15/74 (20%) of sera from MF negative people had IgG antibodies to this antigen. In addition, antibodies to OC 3.6 often preceded the onset of skin snip positivity in Pendie. In contrast, IgG antibodies to OC 3.6 and OC 9.3 were rarely seen in children born during the 15 years since transmission was interrupted by vector control in Niarba. These encouraging results suggest that antibody assays based on OC 3.6 and OC 9.3 may be valuable tools for surveillance of onchocerciasis and also for monitoring the efficacy of control programmes.     

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.     

Induction of histamine release in parasitized individuals by somatic and cuticular antigens from Onchocerca volvulus.

The host immune response in onchocerciasis is believed to contribute to the clinical manifestations of infection. Mazzotti and chronic inflammatory reactions might be mediated by mechanisms involving specific IgE and reactivity of mast cells and basophils to the parasite antigens. In this report, we show that Onchocerca volvulus antigens are capable of inducing histamine release. Three types of extracts were prepared from the parasite: soluble total, surface, and cuticular collagen. Soluble extracts released histamine in all individuals with onchocerciasis at significantly higher levels (P < 0.05) than those found in endemic controls, but similar levels to those found in patients with mansonellosis. However, cuticular collagen induced significantly (P < 0.01) higher histamine release in patients with onchocerciasis than in those with mansonellosis. No reactivity against human type IV collagen was observed. Implications derived from the presence of sensitized basophils in the pathogenesis of onchocerciasis are discussed.     

Identification of radioiodinated cuticular proteins and antigens of Onchocerca gibsoni microfilariae

The filarial parasite of cattle, Onchocerca gibsoni, has been used to establish procedures of antigen identification with a view of applying these techniques to studies on human filarial parasites. Emphasis has been placed on methods suitable for use with small numbers of parasites. Microfilariae (mf) of O. gibsoni were extracted from nodular worms, purified and 125I-labeled using IODO-GEN in solid-phase. Radioactivity was shown to be confined to the cuticle of sectioned mf using the technique of electronmicroscope autoradiography. Radiolabeled mf were analysed by two-dimensional gel electrophoresis. Autoradiographs of 125I-labeled proteins of O. gibsoni mf were relatively complex, there being at least 32 proteins ranging in molecular weights from 20,000 to 120,000 and displaying considerable charge heterogeneity. Evidence was obtained that at least the major serum proteins of the host, albumin or immunoglobulin, were not absorbed on the surface of these uterine mf and detectable in the labeled surface protein patterns. Sera from infected cattle immunoprecipitated 5 labeled proteins from a Triton X-100 extract of 125I-labeled mf. Sera from either of two calves which had been given multiple injections of mf subcutaneously, and which had no detectable skin mf, recognised 6 additional proteins in this extract as well as 3 of the proteins recognised by sera from infected cattle.     

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.     

Small-scale trials of six drugs against Onchocerca volvulus

Six drugs in common use for the treatment of parasitic infections of man were given to 18 adult patients suffering from onchocerciasis. None of the six (metronidazole, tinidazole, mebendazole, trichlorophone, oxamniquine and pyrantel pamoate) showed any evidence of substantial activity against the microfilariae or adult worms of O. volvulus. The mean reduction in skin microfilarial counts a week after drug treatment (a measure of microfilaricidal action) was highest in patients treated with trichlorophone (47.0%) and mebendazole (40.0%). The rate of build-up of microfilariae over a follow-up period of 24 months after treatment with the drug under test followed by DEC (a measure of macrofilaricidal action) was slowest in the groups treated with metronidazole and trichlorophone (22.9% and 27.0% of the pre-treatment counts respectively). These results fall short of those expected of drugs with potential value in the treatment of onchocerciasis.     

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.     

Diagnosis of Onchocerca volvulus infection in Guatemalan children

Nodule examination, the Mazzotti test (a provocative challenge with diethylcarbamazine), and the enzyme-linked immunosorbent assay (ELISA) were compared for diagnostic efficacy in children who were born in an area endemic for onchocerciasis in Guatemala and were skin-biopsy negative for microfilaria of Onchocerca volvulus. Four groups of children under 16 years of age were formed based upon the possible combinations of nodule positivity or negativity and ELISA positivity or negativity. Only those children with a positive (greater than or equal to 1:160) ELISA titer had a positive Mazzotti test (9 of 20 tested), regardless of the presence or absence of nodules. These results suggest that the current ELISA serology is highly sensitive for low density infections, and should be considered in surveys for the incidence of new Onchocerca infections.     

Chemotaxis of human granulocytes toward microfilariae of Onchocerca volvulus

Summary Migration of human peripheral blood granulocytes in response to microfilariae of O. volvulus was demonstrated using modified Boyden chambers. Granulocyte migration was significantly enhanced when microfilariae were preincubated with heat-inactivated immune serum (ΔIS), then added to a fresh serum source (P <0.025). This effect was not seen when microfilariae were incubated in medium alone, in ΔIS alone, in ΔIS plus C4-deficient guinea-pig serum, or in fresh serum alone. There was no significant difference between the response of cells from O. volvulus-infected donors and that of cells from normal volunteers. Likewise, there was no significant difference between the migratory response seen toward nodule- versus skin-derived microfilariae. These results suggest that the host inflammatory response to O. volvulus microfilariae is mediated in part by chemotactic factors generated by antibody and complement interaction with the organism and, furthermore, that these factors are product(s) of classical complement pathway activation.     

Transmission of Onchocerca volvulus by secondary vectors in Guatemala

A comprehensive study of the transmission of Onchocerca volvulus at 4 locations in Guatemala with different prevalence rates of onchocerciasis included observations on potential secondary vectors, the most prevalent of which were Simulium metallicum, S. callidum, and S. gonzalezi. Filariae encountered in S. metallicum were primarily of a Dipetalonema-like species, but third-stage larvae indistinguishable from O. volvulus were found in 4 flies of this species. Our findings suggest that O. volvulus may occasionally be transmitted by S. metallicum, but such transmission is likely limited to areas having both a high parasite prevalence maintained by S. ochraceum and a relatively high host-seeking density of S. metallicum. Two third-stage larvae that could not be differentiated from O. volvulus were found once in S. gonzalezi; however, transmission by this species appears to be inconsequential.     

Emergence of Onchocerca volvulus from skin mimicking Dracunculiasis medinensis

We describe 11 cases of suspected Dracunculus medinensis infection in which the worm recovered was identified as Onchocerca volvulus. Identification was based on morphology of the examined specimen.     

Isolation of living adult Onchocerca volvulus from nodules.

A technique for the isolation of adult Onchocerca volvulus from excised onchocercomata is described. The nodules are incubated in medium 199 containing 1-5 mg collagenase and 0.2 mg gentamicin per ml for 6-48 hours in a waterbath at 30-37 degrees C. A proportion of the worms can be isolated alive.     

Characterization of an Onchocerca-specific DNA clone from Onchocerca volvulus

A genomic library of a savanna isolate of Onchocerca volvulus was screened to detect recombinant plasmids containing highly repeated DNA sequences of this parasite. Four recombinant plasmids were identified which hybridized specifically to Onchocerca DNA, but not to DNA from humans, black flies, Brugia malayi, B. pahangi, or Wuchereria bancrofti. The recombinant plasmids had a low level of homology to Dirofilaria immitis. All recombinant plasmids contain related DNA sequences based on Southern hybridization analysis. Sequences related to these recombinant plasmids are present in different geographic isolates of O. volvulus and O. ochengi, an animal parasite. Two of the recombinant plasmids contain sequences also found in O. lienalis. One recombinant plasmid, puOvs3, has been characterized in detail, including DNA sequence determination. Radiolabeled puOvs3 is able to detect 100 pg of genomic DNA isolated from O. volvulus worms from both savanna and forest regions. It can differentiate O. volvulus from O. ochengi by Southern blot analysis.