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PubMed1998

Detection of Onchocerca volvulus DNA in pools of wild-caught Simulium ochraceum by use of the polymerase chain reaction.

The presence of Onchocerca volvulus DNA in experimentally infected flies can now be detected by use of the PCR, so that, for example, one infected Simulium damnosum can be detected in a pool of 100 uninfected flies or one S. ochraceum can be detected in pools of 20-40. As this PCR technique is specific for O. volvulus, the results are not confounded by the presence of other, unimportant, Onchocerca species, and the technique could replace time-consuming, manual dissection of flies. In 1996 and 1997, pools of 16-21 Simulium ochraceum were tested by the PCR technique. These flies had been collected biting man, between 1992 and 1994, from two hyperendemic coffee estates (fincas) in Guatemala, and stored in commercial (95%) ethanol. Collections at finca Buena Vista (869 flies in 52 pools) were made 1-2 weeks and 46 weeks after 45% of eligible subjects had been treated with ivermectin for the first time. At finca El Brote, collections (360 flies in 18 pools) were made 13 weeks before and 7 weeks after 97% of eligible subjects had received their first treatment. DNA was easily recovered from simuliids that had been stored in ethanol for up to 4 years. Of the nine pools of flies with visible blood collected at Buena Vista, each of 20 flies, eight tested positive for O. volvulus DNA. In flies without blood, 13 of 22 pools collected at Buena Vista just after treatment tested positive, whereas there were 14 positives in 22 pools taken 46 weeks later (P > 0.05). At El Brote, nine of 10 pre-treatment pools were positive, compared with three of eight taken 7 weeks post-treatment (P = 0.04), indicating that the treatments in this finca had reduced infection in the vector, and possibly transmission, by about 60%. A sub-sample of Buena Vista flies was divided into 19 sets of three separate sub-pools containing heads, thoraces and abdomens. Three pools of heads alone were positive, and had corresponding pools of positive abdomens. Three positive pools of thoraces had negative corresponding pools of heads and abdomens. These results show that PCR can be used to determine the prevalence of O. volvulus DNA in wild-caught S. ochraceum. As the infection rates observed were higher than expected from dissections reported by other workers, PCR-determined rates may not be directly comparable with traditional parameters based on the dissection of flies to reveal O. volvulus larvae.

Animals↗
PubMed1993

Ultrastructural observations on the nervous system and the sensory organs of the infective stage (L3) of Onchocerca volvulus (Nematoda: Filarioidea).

A detailed morphological investigation of the sensory organs and the nervous system of the third juvenile stage of Onchocerca volvulus was performed at the ultrastructural level. A complex system of different receptor cells is found at the anterior and posterior end of this developmental stage. The eight papillae are arranged in two concentric circles consisting of two types of morphologically different receptors. Accessory nerve processes end free in the tip of the head. The paired amphids contain nine dendritic processes and accessory axons are seen in the surrounding cells. The basic structure of the amphids and of the circumoesophageal nerve ring is similar to that of other filarial nematodes. Two presumably neurosecretory cells are associated with the nerve ring. The reticular cytoplasm of these cells merges with the rough-surfaced endoplasmic reticulum of the lateral hypodermal chord. The paired phasmids at the posterior end of the developmental stages consist of single modified cilia that are embedded in an electron-dense mass. The receptor cell has access to the outside by a channel ending with a cuticular pore.

Animals↗
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↗
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↗
PubMed1997

Localization of T and B cell stimulating domains of the immunodominant 33-kDa protein of Onchocerca volvulus (Ov33).

The localization of T and B cell epitopes on a well characterized 33-kDa protein of the filarial nematode Onchocerca volvulus (Ov33) was studied using peripheral blood mononuclear cells (PBMC) and sera from a total of 52 onchocerciasis patients with the generalized form of infection. A proportion of the PBMC samples proliferated in response to recombinant Ov33-GST fusion protein and to fusion free Ov33-6xHis. Proliferative responses of patient PBMC to seven truncated Ov33-6xHis polypeptides and to three synthetic peptides revealed at least one major and two minor T cell epitopes in the protein. The dominant T cell stimulating domain was localized between amino acids 113 and 143. ELISA studies with the Ov33-GST fusion protein revealed that patient sera contained Ov33-specific IgG1, IgG4, IgE, and IgM antibodies. Analysis of the IgG4 response with 10 truncated Ov33 polypeptides identified four B cell stimulating domains in the N-terminal, central, and C-terminal region of the molecule. The B cell domain recognized by the majority of sera was localized between amino acids 113 and 143. The data indicate that this region of the protein is the major T and B cell stimulating domain of Ov33 and might be relevant for vaccine development and for improved immunodiagnosis of onchocerciasis.

Adolescent↗
PubMed2003

Impact of parental onchocerciasis and intensity of transmission on development and persistence of Onchocerca volvulus infection in offspring: an 18 year follow-up study.

This study analysed the impact and the extent by which parental Onchocerca volvulus infection, intensity of transmission of O. volvulus infective 3rd-stage larvae (L3) and anthropometric factors may influence the acquisition, development and persistence of O. volvulus infection in offspring. A total of 15290 individuals in 3939 families with 9640 children were surveyed for microfilariae of O. volvulus, and prevalence and level of O. volvulus infection in children aged 0 to 20 years from infected and non-infected parents were followed longitudinally for 18 years. Children from O. volvulus-infected mothers had not only a substantially higher risk to become infected; they also acquired infection earlier in life and developed higher infection levels. Multiple logistic regression analysis showed that maternal O. volvulus infection and children's age are the predominant predictors for patent O. volvulus infection, while the intensity of transmission, measured by the annual transmission potential (ATP) of O. volvulus L3, was less decisive. Longitudinal follow up of children showed that during vector control activities by the Onchocerciasis Control Programme (OCP) and in low-level transmission areas, infection persisted at higher levels in children from O. volvulus-positive mothers. In summary, the dominant risk factor for children to become infected is maternal onchocerciasis, and also age-associated factors will strongly impact on the development of patent O. volvulus infection in offspring.

Adolescent↗
PubMed1988

Preparation and shipping of Onchocerca volvulus material.

Instructions are provided for selection of nodules, isolation of female Onchocerca volvulus and shipping to obtain suitable, intact and live worms for drug-screening and metabolic studies in vitro as well as material for isolation of enzymes, receptors, antigens etc.

Animals↗
PubMed1989

Onchocerca volvulus recombinant antigen: physical characterization and clinical correlates with serum reactivity.

An Onchocerca volvulus cDNA clone expressing epitopes found in adult and larval parasites, designated lambda RAL-2, was derived from a 1,000-base message present in adult O. volvulus, which encodes a protein with an apparent molecular weight of 17,000. This protein does not appear to be extensively posttranslationally modified. Serum samples from 52 individuals exposed to O. volvulus were examined for antibodies recognizing the lambda RAL-2 recombinant antigen; 77% produced such antibodies. In addition, individuals producing antibodies recognizing the recombinant antigen were significantly less likely to develop some aspects of ocular pathology associated with O. volvulus infection than were individuals who did not do so. These results suggest that recombinant antigens such as that produced by lambda RAL-2 may be useful in attempts to understand the mechanism of O. volvulus-induced ocular pathology.

Adult↗
PubMed1997

Onchocerca volvulus: immunohistochemical and immunoelectron microscopical distribution of a polyamine oxidizing enzyme.

We studied the distribution of a polyamine oxidizing enzyme (PAO) in Onchocerca volvulus and other nematode parasites by immunohistochemistry and electron microscopy with immunogold technique using a polyclonal antiserum raised against purified PAO from Ascaris suum. In adult O. volvulus the protein was localized in the outer zone and the area of the basal labyrinth of the hypodermis and occasionally in the outer zone of the uterine epithelium. Further, the fluid in the body cavity was strongly stained. No specific labelling was observed in the cuticle, muscles, epithelia of intestine, ovaries, testis and vas deferens or in sperm, oocytes and embryos. Third-stage larvae of O. volvulus in Simulium soubrense showed strong staining; the same was observed in Anisakis sp. larvae, where the inner and outer zone of the hypodermis were strongly labelled. All mature, intact and dead microfilariae in nodules, skin and lymph nodes were well stained and it was possible to show that the cytoplasm of the hypodermal cells, but not the mitochondria, nuclei or other organelles of muscle cells, was preferentially labelled by immunogold particles. Investigation of adult A. suum presented specific labelling of the hypodermis, but the basal labyrinth was more strongly marked than the outer zone.

Animals↗
PubMed2001

Early human infection with Onchocerca volvulus is associated with an enhanced parasite-specific cellular immune response.

The immune response after early exposure to or infection with Onchocerca volvulus was investigated in an autochthonous focus caused by the migration of infected persons to a previously unaffected area in Ecuador. Peripheral blood mononuclear cell (PBMC) proliferative and cytokine responses (interferon [IFN]-gamma and interleukin [IL]-5) to filarial antigens were measured in 14 subjects with serologic evidence of exposure and in 7 subjects with evidence of dermal microfilarial DNA and were compared with responses in 43 subjects with chronic O. volvulus infections. PBMC proliferative and cytokine responses (IFN-gamma and IL-5) to parasite antigens were elevated in the early exposure/infection group, compared with those in the chronic infection group. Addition of an IL-10-neutralizing antibody to filaria antigen-stimulated cultures resulted in significantly elevated proliferative responses in the chronic infection group. The findings suggest that early exposure and early parasite patency are associated with a vigorous cellular response, but, as infections become chronic, the cellular response becomes down-regulated, partly through an IL-10-dependent mechanism.

Adolescent↗
PubMed2005

Characterization of a novel filarial serine protease inhibitor, Ov-SPI-1, from Onchocerca volvulus, with potential multifunctional roles during development of the parasite.

A novel filarial serine protease inhibitor (SPI) from the human parasitic nematode Onchocerca volvulus, Ov-SPI-1, was identified through the analysis of a molting third-stage larvae expressed sequence tag dataset. Subsequent analysis of the expressed sequence tag datasets of O. volvulus and other filariae identified four other members of this family. These proteins are related to the low molecular weight SPIs originally isolated from Ascaris suum where they are believed to protect the parasite from host intestinal proteases. The two Ov-spi transcripts are up-regulated in the molting larvae and adult stages of the development of the parasite. Recombinant Ov-SPI-1 is an active inhibitor of serine proteases, specifically elastase, chymotrypsin, and cathepsin G. Immunolocalization of the Ov-SPI proteins demonstrates that the endogenous proteins are localized to the basal layer of the cuticle of third-stage, molting third-stage, and fourth-stage larvae, the body channels and multivesicular bodies of third-stage larvae and the processed material found between the two cuticles during molting. In O. volvulus adult worms the Ov-SPI proteins are localized to the sperm and to eggshells surrounding the developing embryos. RNA interference targeting the Ov-spi genes resulted in the specific knockdown of the transcript levels of both Ov-spi-1 and Ov-spi-2, a loss of native proteins, and a significant reduction in both molting and viability of third-stage larvae. We suggest the Ov-SPI proteins play a vital role in nematode molting by controlling the activity of an endogenous serine protease(s). The localization data in adults also indicate that these inhibitors may be involved in other processes such as embryogenesis and spermatogenesis.

Amino Acid Sequence↗
PubMed1996

Human isotype antibody responses to an Onchocerca volvulus glutathione S-transferase.

Human isotype specific antibody responses to a recombinant pi-class glutathione S-transferase (Ov24) from Onchocerca volvulus were assessed by ELISA, using a large and well-characterized bank sera (n = 238) from an hyper-endemic area of moderate intensity from Sierra Leone. IgG1, IgG4 and IgA responses, but neither IgG2 nor IgE response, to Ov24 were detected in infected subjects. The relationships between Ov24 antibody levels and skin microfilarial density, number of nodules, age, sex, eosinophil counts and clinical sign of reactive and chronic pathology were analysed using Pearson's correlation coefficient. Significant correlations between both IgA and IgG3 antibody levels and age were found (P < 0.01). Although no firm conclusions could be drawn from this study sample regarding the relationships between antibody levels and parasite load or clinical status, a negative correlation (P = 0.06) between Ov24 IgG3 antibody levels and microfilarodermia was found.

Adolescent↗
PubMed1991

The embryogenesis of Onchocerca volvulus over the first year after a single dose of ivermectin.

Adult Onchocerca volvulus, extracted from nodules before, and at intervals of two weeks to 12 months after, a single 150 micrograms/kg dose of ivermectin, were examined longitudinally and by sequential transverse sections. The mean number of male worms per nodule fell, and the proportion of nodules with no male worm rose, within two weeks of ivermectin and remained so for 12 months. In female worms, at intervals after ivermectin, the percentages of the length of the lower genital tracts occupied by embryos at each stage of development, or by degenerating ova, embryos and microfilariae (mfs), were recorded: (a) in un(re-)inseminated worms whose original embryogenesis was continuing and in those in which it was completed; and (b) in worms, reinseminated post-ivermectin, in which a new embryogenesis had begun. The results indicated that: (a) the time needed for the zygotes of O. volvulus to develop to mfs is 8-12 weeks; (b) nearly 40 percent of females had not resumed mf production by 12 months after treatment; (c) many intrauterine mfs had not degenerated within the first two weeks of ivermectin; (d) some of the last embryos to mature to mfs did not degenerate but accumulated temporarily in the anterior uteri 8-16 weeks after ivermectin.

Animals↗
PubMed1985

Studies on the protein and antigen composition of individual female Onchocerca volvulus after collagenase digestion.

The composition of proteins and antigens of female Onchocerca volvulus from one focus of transmission was studied by SDS-PAGE and immunoblotting. Worms that had been exposed to collagenase digestion of onchocercomata for different periods of time and parasites of different age and status of reproduction were tested. Some O. volvulus antigens were found to be sensible to prolonged digestion (molecular weights: 18 KD, 21 KD, 24 KD) but the majority of the antigens was stable up to 64 hours of incubation at 34 degrees C. The composition of proteins and antigens varied with the age and the status of reproduction of the worms. Slight differences between individual filariae were found, even when worms of comparable age and status of reproduction were tested that had been exposed to nodule digestion for comparable time.

Animals↗
PubMed1989

Cloning and characterization of an Onchocerca volvulus specific DNA sequence.

A cloned sequence, pOvs134, was isolated from a genomic library prepared from Onchocerca volvulus of savanna origin in the plasmid pUC9. pOvs134 hybridizes to all the geographic isolates of O. volvulus tested from both the New and the Old World, but not to the species Onchocerca gibsoni, Onchocerca gutturosa, Onchocerca ochengi, Onchocerca cervicalis, the filarial parasites Brugia malayi, or Dirofilaria immitis, nor to human or simuliid DNA. As little as 250 pg of DNA can be detected on a dot blot hybridization, suggesting that pOvs134 is sensitive enough to detect a single third stage larva. DNA sequence analysis of the inserted DNA of pOvs134 revealed that it consisted of twelve examples of a 149-bp repeat. The sequence of this repeat is strikingly similar to that of two O. volvulus genomic clones previously described, one of which has been reported to be specific for forest form O. volvulus, and one of which hybridizes to genomic DNA of several species of Onchocerca. These results suggest that the 149-bp repeat sequence is highly repeated in the genome of O. volvulus, and that variants of this repeat with different specificities exist.

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↗
PubMedDate not supplied

Comparison of the sensitivity of different geographical races of Onchocerca volvulus microfilariae to ivermectin: studies in vitro.

This study was designed to provide baseline information on the sensitivity of 4 geographical isolates of Onchocerca volvulus microfilariae (mf) (Ghana forest, Ghana savanna, Cameroon forest and Guatemala) to ivermectin, and to develop an in vitro system with which to examine parasites for the possible development of drug resistance. Drug effects were best visualized in the presence of monkey kidney (LLCMK2) feeder cells in the culture system (MEM medium+20% serum), since mf maintained in the absence of cells declined in condition rapidly. Incubation of Ghana forest mf (+cells) in ivermectin (10(-5)-10(-10) M) caused a decrease in motility index (MI) scores in a concentration-dependent fashion; drug effects could be observed as early as 6 h, but cultures maintained for up to 8 d showed greater differences between control and drug groups with increasing time. All 4 O. volvulus isolates and O. lienalis (bovine) were compared for their response to ivermectin (10(-7) M): O. lienalis mf were significantly more sensitive (78%) reduction in MI scores on day 8) than the O. volvulus isolates (33.4-47.7% reduction). O. volvulus microfilariae ex utero generally displayed lower levels of motility and were slightly less inhibited by ivermectin than were skin mf. The in vitro system described can distinguish between the populations of mf studied on the basis of differing MI responses to ivermectin and, when combined with assays to test the infectivity of mf to blackflies following exposure to drug, will provide methods with which to examine parasites for the possible development of resistance.

Animals↗