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PubMed1990

An improved method of examining adult Onchocerca volvulus worms.

Adult Onchocerca volvulus worms were extracted from alcohol-fixed nodules by collagenese digestion. The worms were stained in Mayer's haematoxylin and brought up into glycerol. In this viscous fluid the stained and cleared worms could be unravelled in sequential lengths of 4-6 cm and mounted in glycerol on slides for longitudinal examination of their structure. The worm lengths were then cut into small pieces, 0.5 cm long, which were implanted sequentially into slits in a brick-shaped block of formalin-fixed calf's brain (white matter) also in glycerol. The brain blocks were then processed in paraffin wax and cut as 6 microns histological sections, enabling transverse sections of the worm, stained as required, to be examined in sequential order.

Animals↗

PubMed1989

[Effects of diethylcarbamazine and ivermectin on the mobilization of microfilariae of Onchocerca volvulus].

Microfilaria of Onchocerca volvulus are localized in superficial dermis. However, they are sometimes noticed in the blood and urines, when there is an important infestation. Diethylcarbamazine (DEC) bring on an increase of microfilaria, successively in the blood and after, in the urines. This study of 30 patients, treated in double blind by placebo, DEC and ivermectin, a new molecule with spectacular action on this filariasis, allowed to compare effect of each one on apparition of microfilariae in the blood and urines. Ivermectin bring on very important increase of microfilaremia without microfilaruria, unlike diethylcarbamazine. This establishment bring to discuss very different physiologic mechanisms between them. It should have an incidence on diagnosis and treatment of this dangerous parasite.

Adult↗

PubMed1981

A diagnostic skin test for Onchocerca volvulus infection.

Onchocerca supernatant (OS) was prepared by a technique permitting live microfilariae to migrate from nodule tissue through agar gel into sterile Hanks balanced salt/Penicillin-Streptomycin solution where they metabolized. The OS, after dialysis, was passed through Seitz viral filter and either concentrated or lyophilized. Using rabbit antiserum in immunodiffusion and immunoelectrophoresis tests, microfilariae proteins and also human protein were detected in out OS. No common antigens were found between this and somatic extracts of Loa loa, O. gutturosa, O. volvulus, L. carinii, D. immittis and A. lumbricoides. 125I labelled OS was purified by passage through protein A column and then through immunosorbent column of horse anti-human serum linked to CNB-activated sepharose 4B. Autoradiography, after sodium dodecyl sulphate polyacylamide slab gel eletrophoresis of purified OS, showed 10 protein bands in the molecular range 10,000 to 125,000. Skin prick tests with OS, shown not to be contaminated with Hepatitis B antigens, elicited immediate hypersensitivity reaction. Using our criteria, positive reactions were seen in 81% of proven onchocerca cases and only occasionally in Loasis 4.5%, ascaridiasis 13.5% or healthy controls 2.4%. The poor skin reactivity to OS in loasis was not due to immunosuppression as these patients, when also infested with ascaris, reacted just as well as onchocerca patients with ascaris to skin prick test using somatic extracts of ascaris.

Adolescent↗

PubMed1976

Preliminary identification and characterization of antigen extracts from Onchocerca volvulus.

Sequential extraction of Onchocerca volvulus from skin nodules with buffers of different ionic strength resulted in a mixture of antigens which reacted in double immunodiffusion with sera from proven cases of onchocerciasis, the number of precipitation lines varied from 1 to 5. Triton X-100 detached mainly cationic antigen proteins. It reduced the molecular size of many antigenic proteins, but did not effect their antigenicity. Labelling of the extracted proteins with high specific activity 125I did not destroy much antigenicity. Strong cross-reactivity between Onchocera volvulus extracts and Onchocerca gutturosa and Litomosoides carinii was observed, less with extracts of Necator americanus and Ascaris suum, and none with Echinococcus multilocularis. Sera from proven cases of wuchereriasis, toxoplasmosis and echinococcosis rarely contained antibodies against O. volvulus. Isoelectric focusing, immunoelectrophoresis and gel filtration of the O. volvulus extracts indicated a variety of antigens. Preliminary attempts were made to purify the antigens using a solid-phase immuno-adsorbent prepared by coupling purified onchocerciasis IgG to CNBr-activated Sepharose.

Antigens↗

PubMed1999

[Proteolytic activity of adult worm extracts of Onchocerca volvulus].

Studying proteolytic activity of Onchocerca volvulus (nematode causing "river blindness") shows that it is able to digest a variety of substrates such as: azoalbumine, azocoll and elastin-orcein with specific activity of 0.28, 0.57 and 1.48 mg/hour/mg of extract respectively. These enzymes are active at various pH such as pH 5.0, 8.0 and 10.0 with highest activity at pH 8.0. The effect of specific inhibitors and activators indicates that the extract might contain serine, metallo and thyoproteases. The electrophoresis of the extract on a polyacrylamide gel copolymerized with gelatin shows many proteins with enzymatic activities with molecular weight of 16.6, 43.6, 45.7, 56.2, 60.2, 61.6 and 63.1 KD respectively. The Onchocerca volvulus worm contains proteases of various enzymatic activities: a non specific activity on protein such as on azoalbumin and specific activities on collagen and elastin. These enzymes could play an important role in the survival of parasites in human hosts.

Albumins↗

PubMed2001

Development of a recombinant antigen vaccine against infection with the filarial worm Onchocerca volvulus.

Efforts to control Onchocerca volvulus, the etiologic agent of river blindness, have been limited to vector control and drug treatment to eliminate microfilariae, with no means available to prevent infection. The goal of this study was to develop a vaccine against this infection using recombinant antigens that are expressed in the early larval stages of the parasite. Five recombinant antigens, Ov7, Ov64, OvB8, Ov9M, and Ov73k, were identified by screening adult and larval cDNA libraries with antibodies from immune humans, chimpanzees, or rabbits. When mice were immunized with the five individual recombinant antigens, statistically significant reductions in parasite survival were induced in mice immunized with Ov7, OvB8, or Ov64, when administered in alum but not when injected in Freund's complete adjuvant (FCA). Live larvae recovered from control and immunized mice were analyzed to determine their developmental stages. A decrease in the percentage of larvae molting from the third stage to the fourth stage was observed with mice immunized with Ov7, Ov64, or OvB8 in alum but not with mice immunized with Ov9 and Ov73k or with mice immunized with any of the five antigens in FCA. Mice immunized with a cocktail of the three protective antigens developed protective immunity equal to that seen with mice immunized with individual antigens. This study has identified, for the first time, three recombinant antigens capable of inducing protective immunity to O. volvulus. Furthermore, since the antigens functioned with alum as the adjuvant, this vaccine could potentially be used clinically to prevent river blindness in humans.

Adjuvants, Immunologic↗

PubMed1992

The actin genes of Onchocerca volvulus.

The genome of Onchocerca volvulus was found to contain 2 actin gene classes (called 1 and 2) of 2 genes each. The 4 genes are located in 2 clusters (called A and B), each containing a gene class member. Five short introns of 122-207 bp occur within each gene. The sequences of the fourth intron and the 5' and 3' untranslated regions of all the 2 gene classes are completely different even though their coding regions share 95% identity. Mature transcripts from the actin genes have the nematode spliced leader (SL) at their 5' ends. One actin cDNA was found to be derived from an actin pre-mRNA which locks both the 5 introns and the 5' SL, suggesting that in at least some transcripts cis-splicing is completed before trans-splicing occurs.

Actins↗

PubMed1985

Serum immunoglobulin E levels in onchocerciasis: the development of a radioallergosorbent test for Onchocerca volvulus infection.

The effects of Onchocerca volvulus infection on immunoglobulin levels have been studied in subjects from three localities with different onchocerciasis prevalence rates. Infestation by O. volvulus provoked a 200-500% rise in total serum IgE levels. Immunoglobulins G, M. and A were increased less dramatically. To study further the IgE response, radioallergosorbent test (RAST) systems were developed using total extract of O. volvulus and Onchocerca supernatant (excretory-secretory) antigens. Both antigen preparations reacted with more than 80% of the onchocerciasis sera from the hyperendemic village, but the reactions were not proportionate to skin microfilarial density. There were also positive reactions with sera of some individuals whose skin biopsies showed no microfilariae, indicating prior exposure to the parasite. European control sera tested under similar conditions reacted only slightly in the RAST system. The developed RAST systems may thus be used for determining prior exposure to O. volvulus.

Cameroon↗

PubMed1977

Intracytoplasmic bacteria in Onchocerca volvulus.

Ultrastructural studies on Onchocerca volvulus disclosed intracellular organisms within the lateral chords of adult worms and of the larval stages. In the females the organisms were also present in the oogonia, oocytes, developing eggs and microfilariae. The organisms, found within vesicles of host (filarid) membrane and limited to the cytoplasm of infected cells, appeared to have a developmental cycle consisting of three morphologically distinct forms: a small spheroidal form up to 0.3 micronm in size, a bacillary form up to 1.5 micron7 in length and 0.7 micronm in diameter, and a third form, intermediate in size between the former and the latter, characterized by a dense inclusion. The intravesicular location and the developmental cycle consisting of three distinct forms are the two characteristics which suggest that these organisms are more similar to the chlamydiae than to the rickettsiae, in spite of their being transovarially transmitted. The significance of these findings with respect to the host-parasite relationship and pathogenesis of onchocerciasis is presently unknown and will require further study.

Animals↗

PubMed1978

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.

Antigens↗

PubMed1996

Structural and molecular specificity of antibody responses in mice immune to third stage larvae of Onchocerca volvulus.

Immunization of mice with irradiated Onchocerca volvulus infective stage larvae (L3) has been demonstrated to confer protection against challenge infections with these larvae. Additionally, cytokine level measurements and cytokine depletion studies have shown that both IL-4 and IL-5 are important in generating a protective immune response against O. volvulus challenge infections, thus suggesting a dependency of protective immunity on IgG1, IgE and/or eosinophils. In the present study, we examined the humoral responses of immunized mice to O. volvulus L3 antigens. ELISA measurements of total serum antibody levels indicated that IgE was the only antibody isotype elevated in mice immunized with O. volvulus L3. IgM from immunized mice was the only isotype that recognized surface antigens on intact O. volvulus L3. IgG1, IgG3, IgE and IgA recognized internal parasite antigens on O. volvulus L3 frozen sections. Western blot analysis of L3 proteins showed that in serum from mice immunized with O. volvulus L3 IgG1, IgG2a/2b, IgA, and IgE, as well as IgM, recognized unique L3 proteins. Antibodies in serum from L3 immunized mice were able to detect O. volvulus adult antigens in a pattern similar to the recognition found in O. volvulus L3. Some L3 antigens were shared by adults, while other antigens were L3 specific. The ELISA, immunohistochemistry and Western blot findings thus demonstrate a complex pattern of antigen recognition of parasite antigens by antibodies found in mice immune to the L3 of O. volvulus.

Animals↗

PubMed2000

Regional genetic variation in the major sperm protein genes of Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea).

Onchocerca volvulus and Mansonella ozzardi are two human filarial parasites present in South and Central America. In the Brazilian Amazonia they are found in sympatry, and the lack of clear morphological diagnostic characters in the microfilariae hinders their identification. The major sperm protein (MSP) gene of both species has been sequenced and characterised to determine its potential as a molecular diagnostic character. The length of the MSP gene is different in each species, and this could be used to detect and differentiate them by running the polymerase chain reaction (PCR) product in an agarose gel. Two major gene groups were identified in O. volvulus with a genetic distance of 6% between them. In M. ozzardi only one major group of genes was observed. The high similarity between the protein amino acid sequence of both filarial species confirms that the MSP has been highly conserved through nematode evolution.

Animals↗

PubMed1997

PCR and DNA hybridization indicate the absence of animal filariae from vectors of Onchocerca volvulus in Uganda.

In order to identify Onchocerca volvulus larvae from vectors, DNA of filaria larvae from dissected blackflies was isolated, and a 150-bp long tandemly repeated DNA sequence (0-150), which occurs in many Onchocerca species, was amplified using polymerase chain reaction (PCR). Subsequently, the PCR product was blotted onto a nylon membrane and hybridized with DNA probes specific for O. volvulus or Onchocerca ochengi. Filaria larvae from 395 infected Simulium neavei were examined and 259 samples produced detectable PCR products. Among these samples, 239 (92%) reacted with an O. volvulus-specific oligonucleotide. A sample of 69 PCR products was tested using an O. ochengi DNA probe, but all failed to hybridize. Filaria larvae from 64 infected Simulium damnosum, presumably of the cytotypes "Nyamagasani" and "Nkusi" were studied and 0-150 was amplified from 38 samples. From these samples, 35 (92%) hybridized specifically with an O. volvulus probe but none with the O. ochengi-specific DNA sequence. Nonamplified samples were obtained mainly from blackflies that contained only 1 or 2 filaria larvae, and therefore, an insufficient DNA extraction was assumed. It can be concluded that few, if any, filaria species of animal origin were transmitted by S. neavei and S. damnosum s.l. in Kabarole and Kasese districts in Uganda.

Animals↗

PubMedDate not supplied

Tolerance to diethylcarbamazine-medicated salt in individuals infected with Onchocerca volvulus.

The tolerance of Onchocerca volvulus-infected individuals to diethylcarbamazine (DEC)-medicated salt (0.33% w/w) was assessed in 1996 in Tanzania in a double-blind placebo-controlled hospital-based trial involving 4 groups, each of 10 adult males. Groups I and II had O. volvulus microfilariae (mf) only, group III had both O. volvulus and Wuchereria bancrofti mf, and group IV had W. bancrofti mf only. Groups I, III and IV received DEC-medicated salt, whereas group II was a control to group I and received normal cooking salt. Medication was given for 10 days. The most pronounced adverse reactions in groups I and III were mild-to-moderate itching and rash, beginning after 3-4 days and lasting for the remaining medication period. The reactions did not interfere with normal daily activities. By 20 days after the end of medication, adverse reactions had disappeared in all individuals. The low daily dose of DEC had no significant effect on the O. volvulus pre-medication mf geometric mean intensities (GMIs). In contrast, the medication significantly reduced the pre-medication W. bancrofti mf GMIs. The prospects for using DEC-medicated salt for control of bancroftian filariasis in areas where incidental infections with O. volvulus occur are discussed.

Adult↗

PubMed1990

In vitro assessment of the activity of anthelmintic compounds on adults of Onchocerca volvulus.

The viability of adult Onchocerca volvulus and the effect of 12 known anthelmintic compounds on the parasites have been evaluated in an in vitro culture system. Three different parameters, a colorimetric assay, using NADH-dependent reduction of a tetrazolium salt to dark blue formazan by living adult worms, motility indices of male worms and lactate excretion of female worms were used to determine worm viability. The experiments showed that over a short term period of six days the viability of the worms did not decline significantly. The use of males isolated by dissection of whole nodules for the evaluation of drug effects in vitro is preferable to collagenase isolated worms. Mel W, milbemycin a and d, ivermectin, levamisole, CGP 6140 and, to a lesser extent, suramin immobilized male worms or significantly reduced the motility indices at a concentration of 10 microM. The tetrazolium reduction by male worms was not affected by levamisole, whereas the other active compounds demonstrated significant inhibitory effects. Diethylcarbamazine, mebendazole, flubendazole, metrifonate and CGP 20376 had no significant effect on male viability. Comparable activity was seen with the intact female worms isolated by collagenase digestion. Mel W, the milbemycins and ivermectin significantly inhibited tetrazolium reduction, whereas suramin and the other compounds had only slight or no inhibitory effects on female O. volvulus. Although one still has to aim at an improvement of the culture conditions, the in vitro test system using adult O. volvulus provides a basis for further research on potential antifilarial compounds.

Animals↗

PubMed2006

Molecular studies of Onchocerca volvulus isolates from Mexico.

DNA from Onchocerca volvulus from Oaxaca and Chiapas, Mexico were used as templates to amplify members of the O-150 Onchocerca specific repeat sequence family. The resulting PCR amplicons all hybridized with OVS2, an oligonucleotide that has been previously shown to recognize amplicons derived from O. volvulus with 100% sensitivity. However, when PCR products amplified from the O. volvulus specific plasmid pOVS134 were used as a probe, most samples did not hybridize. Similarly, when PCR products amplified from DNA isolated from adult O. volvulus from Oaxaca were used as a probe, amplicons from adult worms from both Oaxaca and Chiapas were recognized, but PCR products from infected black flies from Chiapas were not recognized. Amplicons derived from an adult worm from Chiapas hybridized with PCR products produced from adult parasites from both Oaxaca and Chiapas and to PCR products derived from the DNA of infected black flies from Chiapas. These data, when taken together, suggest that differences exist among the repeat sequence populations of parasites from Oaxaca and Chiapas in Mexico, suggesting that the O-150 repeat sequence family may be a useful tool for biogeographic studies of O. volvulus in the Americas.

Animals↗

PubMed1984

Observations on adult Onchocerca volvulus maintained in vitro.

Adult Onchocerca volvulus were enzymatically isolated with collagenase from excised nodules and kept in TC medium 199 with Hank's salts supplemented with various sera. Male and female worms survived in the culture medium and 10% human serum on average for about 11 days (maximum 28 days) and 14.5 days (maximum 42 days). Up to 4,000 microfilariae were expelled by each female per day, but the production of new oocytes or development of embryos could not be observed in vitro.

Animals↗

PubMed1988

Vector density, parasite prevalence, and transmission of Onchocerca volvulus in Guatemala.

Transmission of Onchocerca volvulus at 4 locations with different prevalences of human onchocerciasis in the Atitlán region of Guatemala is described in relation to vector density and infection rates. The percentages of residents with skin biopsies positive for microfilariae of O. volvulus at these locations were 13.8%, 33.3%, 65.4%, and 89.6%. The following variables associated with transmission were calculated from our observations (the values are presented in an order that corresponds with the above prevalence rates): frequency of third-stage larvae (calculated on an annual basis) in parous Simulium ochraceum, 0, 0.004, 0.005, and 0.004; estimated daily biting density of S. ochraceum, 23, 24, 254, and 1,509 flies per day; and estimated annual infective biting density (based on S. ochraceum), 0, 18, 185, and 1,101 potentially infective bites per year. The frequencies of third-stage larvae are very small compared with those observed in Africa, and suggest that transmission of O. volvulus in Guatemala depends on high vector density. Locations with low, and perhaps tolerable, levels of onchocerciasis (less than 15% of female residents with skin biopsies positive for microfilariae) have mean daily biting densities for S. ochraceum of less than or equal to 24 flies, and infected residents normally have mean microfilarial densities of less than or equal to 3 microfilariae per mg of skin. Stratification of prevalence rates by age group proved useful for assessing current transmission within a village.

Adolescent↗