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PubMed1995

The in vitro culture of Onchocerca spp.: I. Preliminary observations on the development of the second to the third stage larvae.

Second stage larvae of Onchocerca lienalis and O. gutturosa were induced to moult successfully to the third stage in vitro. Following development in their vectors Simulium ornatum s.l. and Culicoides nubeculosus the second stage larvae were kept in a cell free culture system. At least 25% of the second stage larvae of either Onchocerca species completed the moult successfully although no feeder layers had been used. In the absence of CO2 (5% in air) the number of the second stage larvae moulting successfully to the infective stage dropped to 6 and 9% respectively.

Animals↗
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↗
PubMed1997

Production of monoclonal antibodies against excretory-secretory products of adult male Onchocerca gibsoni.

Nine monoclonal antibodies (mabs) have been produced against excretory-secretory products (ES) of adult male Onchocerca gibsoni. These mabs fail to interact with the highly cross-reactive phosphorylcholine (PC) group and with ES of the related rodent filarial parasites Acanthocheilonema viteae and Litomosoides carinii. Eight of the mabs are of the IgG isotype: 1 is an IgM. Three of the mabs, OGMES 4, 9, and 10, were each found by immunoprecipitation/SDS-PAGE analysis of [3H] leucine-labeled ES, to recognize a triplet of polypeptides of molecular weight 120, approximately 210, and approximately 260 kDa. No recognition was observed by any mab when [3H] glucosamine was employed as the radiolabel for ES. Western blotting employing [125I] as indicator system demonstrated that OGMES 7 recognized a molecule of 27 kDa, and OGMES 1, a molecule of approximately 210 kDa, albeit faintly. These mabs may be of value to researchers working on the isolation, characterization, and detection in the bloodstream of Onchocerca volvulus ES.

Animals↗
PubMed1976

Antigenic diversity among Onchocerca volvulus in Nigeria, and immunological differences between onchocerciasis in the savanna and forest of Cameroon.

Onchocerciasis is a remarkably chronic infection and, in West Africa, there are distinct clinical and pathological differences between the disease in the savanna and in the forest. Experiments were carried out to see whether antigenic diversity among Ochocerca volvulus worms might contribute to these features. Extracts of adult worms and or microfilariae were analysed by disc electrophoresis on polyacrylamide gels and showed a remarkable diversity of protein patterns but no consistent savanna/forest or Nigeria/Cameroon differences. Indirect haemagglutination using cross-absorbed sera was used to look for antigenic diversity or identity between different O. volvulus worms. Sera of patients were absorbed with antigens extracted from worms taken from those paitents and then titrated against sheep erythrocytes coated with the same antigens. It was shown that an adult Onchocerca volvulus could be either antigenically identical with, or distinct from, another worm taken from the same patient. In one Nigerian village identity was shown in two instances nad diversity in five. Microfilariae lack some antigens possessed by adult worms. This test has shown consistent savanna/forest differences in worm antigen patterns and antibody responses. In order to explain the pattern of these differences it was necessary to postulate that forest patients possessed antibodies to another worm which had antigens in common with savanna Onchocerca. The relevance of these findings to the pathology and prevention of onchocerciasis is discussed.

Antigens↗
PubMed1998

[Onchocerca volvulus--a rare cause of a mass in children].

The parasite Onchocerca volvulus is well-known in its endemic areas in South and Central America and West Africa. It is transmitted to man by simulium flies and causes systemic infection with skin, lymphatic and ophthalmic manifestations and can cause blindness (river blindness). Treatment with Ivermectin is effective but sometimes there is need for surgical intervention to prevent or treat complications. We describe an 11-year-old girl, a new immigrant from Ethiopia, who had a firm mass in her left thigh, caused by Onchocerca volvulus. It was completely excised. This is a very rare condition in Israel, which must be considered in patients coming from endemic areas.

Animals↗
PubMed1998

Cloning and characterization of cDNAs encoding beta-tubulin from Dirofilaria immitis and Onchocerca volvulus.

Beta-Tubulin is the target for the benzimidazole anthelmintics. Unfortunately, none of these drugs is clinically useful against adult filariae. However, beta-tubulin has been shown to be a target for antibody-based toxicity to Brugia pahangi. We cloned and characterized cDNAs encoding beta-tubulin from 2 filariae, Dirofilaria immitis and Onchocerca volvulus, to explore possible explanations for benzimidazole insensitivity among adult filariae and the likelihood that epitopes of beta-tubulin could be used as antigens for a broad-spectrum filarial vaccine. The proteins predicted by these cDNAs were almost identical to the beta-tubulin previously reported from B. pahangi but were less similar to a beta-tubulin cDNA from Onchocerca gibsoni. We cloned the genomic locus for the O. volvulus beta-tubulin cDNA and compared its organization to the reported genomic loci for beta-tubulin in B. pahangi and O. gibsoni. The comparison reinforces the conclusion that the published O. gibsoni gene is in a different family, possibly the beta2 family previously described in B. pahangi. The substitution of tyr for phe at position 200 of beta-tubulin is associated with benzimidazole resistance. All 4 filarial beta-tubulins are predicted to encode phe at this position, suggesting that filarial beta-tubulin is not inherently insensitive to the benzimidazoles. A monoclonal antibody that recognizes the COOH terminus of B. pahangi beta-tubulin is lethal to this parasite in culture. The COOH terminal region is the most variable among the different isotypes of beta-tubulin and distinguishes mammalian from nematode tubulins. This region is highly conserved in 3 of the filarial beta-tubulins.

Amino Acid Sequence↗
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↗
PubMed2005

Increase in cellular immune responses in Onchocerca-infected cattle after treatment with the microfilaricide, milbemycin.

Lymphocytes from Onchocerca-infected steers treated with the microfilaricide, milbemycin showed increased proliferation when challenged with antigen from Dirofilaria immitis, concanavalin A, tuberculin and tetanus toxoid, compared with untreated animals. This paper confirms that Onchocerca infection induces immunosuppression to filarial and non-filarial antigens. It raises the possibility that filarial-induced immunosuppression may increase the susceptibility to mycobacterial infections and reduce the efficacy of vaccinations and strongly indicates that further research is required.

Animals↗
PubMed1997

The entry of ivermectin and suramin into Onchocerca ochengi nodules.

No currently available drug, which is safe for mass treatment, effectively kills adults of Onchocerca volvulus, the causal agent of onchocerciasis in humans, or of O. ochengi, a cattle parasite used as a model of O. volvulus. Since adults of both of these filarial nematodes are found in well developed nodules, the lack of efficacy of these drugs may be a result of their poor penetration into the nodules. To check if this was the problem, the distributions of the microfilaricide, ivermectin, and the partial macrofilaricide, suramin, in plasma, skin, nodule capsules and nodule contents were determined in cattle naturally infected with O. ochengi in Cameroon. The cattle were treated with either a single, subcutaneous injection of 500 micrograms ivermectin/kg, or with intravenous injections of [14C]-labelled suramin, each of 10 mg/kg, given one a day for 6 days. Concentrations of ivermectin and suramin in various tissues were then assayed by high-pressure liquid chromatography and scintillation counting, respectively. On day 7 post-treatment (pt), suramin concentrations were consistently highest in the nodule, contents and capsule wall (11.0 and 8.9 nCi/g, respectively) and significantly less in skin and plasma (1.2 and 1.4 nCi/g, respectively; P < 0.05). The distribution of ivermectin on day 7 pt was similar, with the highest concentrations in the capsule wall, nodule contents and plasma (58.4 ng/g, 43 ng/g and 48.6 ng/ml, respectively; P > 0.05) and the concentration in the skin (6.4 ng/g) significantly lower than those in the capsule or plasma (P < 0.05). High intra-nodular concentrations of both drugs were maintained for 5-7 days at least and those of ivermectin would be expected to kill nematodes other than filariae. It is apparent that failure of ivermectin and suramin to kill adult Onchocerca spp. is not because the drugs penetrate nodules inadequately.

Animals↗
PubMed1988

Autoantibody induced by experimental ocular Onchocerca infection: identification and characterization of autoantigens.

Hartley guinea pigs were injected with Onchocerca lienalis microfilariae (Mf) as a model for human infection with Onchocerca volvulus. Subconjunctival injection of guinea pigs with O. lienalis Mf results in penetration of Mf to the central cornea, and formation of acute inflammatory reactions resembling those of human onchocerciasis around dead Mf. Sera from these animals contain IgG autoantibodies directed against at least two components of 3M KC1 extracts of guinea pig cornea and liver. Components with apparent molecular weights of 94-99 and 110-120 kilodaltons (KD) were recognized in both cornea and liver extracts, while 100, 74 and 34 kD components were recognized in liver extracts only. Components of 140 kD and 84 kD were recognized in cornea extracts only. The guinea pig liver extract cross-reacted with homogenate antigens from O. lienalis Mf, while the guinea pig cornea extract did not. Autoantibody-mediated inflammatory mechanisms may contribute to immunopathologic processes in onchocerciasis.

Animals↗
PubMed1983

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.

Adolescent↗
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↗
PubMed1979

[The epidemiological significance of neo-natal parasitism with microfilariae of Onchocerca volvulus (author's transl)].

Between April 1977 and June 1978, 214 babies born of 210 mothers infected with Onchocerciasis, were examined at the maternity of Po Hospital (Upper Volta). In four of them (1.9%) dermal microfilariae of Onchocerca volvulus were found during the first week after birth. In two other cases microfilariae were seen in the tissue of the umbilical cord. All specimens from placentas and amniotic fluid gave negative results. The infected babies were reexamined monthly monthly from 2 to 18 months, depending on the cases. One child remained positive until the 6th month follow-up. The base-line examination of 5.757 children less than 2 years old from different villages in the WHO Onchocerciasis Control Programme in the Volta River Basin Area showed that 1% of children less than one year old and 2% of children of 12 to 23 months old were carriers of dermal microfilariae. These results indicate that in meso- or hyperendemic areas children less than one year old and carriers of microfilariae of Onchocerca volbulus may have been infected in utero. The existence of such cases, in an area of vector control, does not prove a residual vector borne transmission.

Burkina Faso↗
PubMedDate not supplied

Onchocerca lesions in moose (Alces alces L.).

Inflammatory discolorated granulomas occurred in 98% of investigated moose, 1.5 years of age or older. In moose calves no lesions were observed. The lesions were mainly located in tendineous tissues. Predilection sites were the insertions of M. sartorius and M. semitendinosus at the tibia. Lesions also occurred in peritendineous and subcutaneous tissues. The lesions were caused by Onchocerca worms. The character of the inflammatory reaction was benign and onchocercal lesions in moose appeared to be of minor importance for the health and mobility of the animals, and also from meat hygiene aspects. The found worm shows similarities regarding morphology and localization to O. tarsicola. As the worm, however, is very common in moose and seems to be well adapted to this animal, the presence of an Onchocerca species or strain specific to moose, can not be ruled out.

Animals↗
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↗
PubMed1994

Protective immunity against Onchocerca volvulus and O. lienalis infective larvae in mice.

Acquired resistance to both Onchocerca volvulus and O. lienalis infective larvae, implanted within micropore chambers, could be induced in mice following immunization with irradiated L3 larvae. In experiments with O. volvulus in BALB/c and BALB/c. By mice, consistent levels of protection (61-75% reductions compared to challenge controls) were achieved with challenge infections of 2 week duration. In DBA/2 mice, levels of protection against O. lienalis were lower and more variable (42-63%): Moreover a 3 week period between challenge and recovery was required before significant reductions in larval recovery became detectable in vaccinated animals. Immunization of CBA or BALB/c mice with O. lienalis microfilariae, or CBA mice with normal or irradiated O. lienalis L3 larvae, failed to induce killing or growth retardation of developing larvae. Preliminary characterization of the effector mechanisms and cytokines associated with protective immunity against O. volvulus infective larvae revealed elevated levels of eosinophils in peripheral blood and within micropore chambers during challenge infections in vaccinated mice. Spleen cells from the same animals stimulated with parasite antigen induced significant levels of IL-5, IL-4 and IFN gamma. These cytokines were barely detectable in antigen stimulated cells from challenge control mice.

Animals↗
PubMed1986

Punctate keratitis induced by subconjunctivally injected microfilariae of Onchocerca lienalis.

An experimental model of human onchocercal keratitis was produced by injecting guinea pigs subconjunctivally with microfilariae (Mf) of Onchocerca lienalis. Actively motile O lienalis Mf spontaneously penetrated the central cornea and produced gray-white midstromal opacities, 0.2 to 0.4 mm in diameter, resembling those of human onchocercal punctate keratitis. Histologically, small foci of eosinophil and mononuclear cell infiltration associated with small pockets of interstitial edema were present in the central corneal stroma, with eosinophil infiltrates in the conjunctiva, episclera, limbus, and ciliary body. The severity of the punctate keratitis was increased by repeated subconjunctival inoculations of Mf. Punctate lesions were not seen following subconjunctival injection of Mf in animals previously hyperimmunized by three subcutaneous injections of Mf. Hyperimmunization may produce an immune response capable of destroying the subconjunctivally injected Mf and preventing their migration into the central cornea.

Animals↗
PubMed1988

Experimental interstitial keratitis induced by Onchocerca volvulus antigens.

To elucidate the pathogenic mechanisms involved in onchocercal sclerosing keratitis in humans, we developed a model of onchocercal interstitial keratitis in guinea pigs. Onchocerca volvulus antigens injected intrastromally into corneas of preimmunized Hartley guinea pigs induced an intense stromal keratitis with corneal edema, neovascularization, and infiltration with acute and chronic inflammatory cells. This reaction subsided after two weeks. Repeated intrastromal injection resulted in an exacerbation of the keratitis and ultimately in residual scarring. These findings are consistent clinically and histopathologically with the chronic interstitial keratitis observed in humans. To define which antigens induce the corneal reaction, O volvulus antigens were separated by molecular sieve chromatography and injected intrastromally. The highest activity was shown to reside in the fraction containing molecules of intermediate molecular weight. This model will be useful in defining O volvulus antigens and their role in sclerosing keratitis as well as in elucidating the immune mechanisms involved.

Animals↗