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PubMed1973

Daytime variation in the density of Onchocerca volvulus microfilariae in human skin.

One aim of this study was to determine whether a corneal-scleral punch biopsy instrument provides skin specimens of sufficiently uniform size so as to eliminate the need to weigh or measure the specimens in order to provide a reproducible index of the density of Onchocerca volvulus microfilariae in the skin. A second aim was to determine whether the density of microfilariae in the skin varies with the time of day. The reports of others on the latter are contradictory. Duplicate skin specimens were taken from 10 people four times during one day with a corneal-scleral punch biopsy instrument. Each specimen was weighed and measured, and the number of microfilariae were counted. Although the specimens were not of uniform weight or surface area, the number of microfilariae per specimen was as good an index of the density of larvae in the skin as the number of microfilariae per mg, or per mm(2) of skin. This may not be true when other methods of procuring skin specimens are utilized. A universally acceptable standard method is advocated. The density of microfilariae in the skin approximately doubled from morning to midday, and began to decline by early evening.

Adult↗
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↗
PubMed1968

The effects of drugs on Onchocerca volvulus. 2. The antimonial preparations TWSb and MSbE.

Antimonial preparations (Pentostam, Neostibosan, stibophen, and tartar emetic) have occasionally been used in the treatment of onchocerciasis without very promising results. The advent of the preparations TWSb (stibocaptate) and MSbE (Friedheim) of allegedly reduced toxicity made it desirable to test them against Onchocerca volvulus.The action of both preparations on the parasites was found to vary from one patient to another, ranging from complete elimination of all parasites in a few cases to no detectable action in others. A microfilaricidal action was detectable in many patients, particularly after treatment with TWSb, which was used at higher doses than MSbE. A lethal or sterilizing action on some or all adult female worms was observed in some patients. However, toxic reactions to the drugs were common and distressing, and often it was necessary to stop treatment on this account. Anorexia, nausea, vomiting and prostration were the most common manifestations, and there was one fatality from coincident yellow fever, which may well have been aggravated by antimony treatment.The uncertain action of these preparations on O. volvulus and the toxic manifestations that accompany their use render them unsuitable for the treatment of onchocerciasis, and it is probable that the effects of antimony on O. volvulus are produced only at or above the normal level of human tolerance.

Animals↗
PubMed1968

The effects of drugs on Onchocerca volvulus. 3. Trials of suramin at different dosages and a comparison of the brands Antrypol, Moranyl and Naganol.

Suramin and Mel W are the only two drugs at present known to be effective against the adult worms of Onchocerca volvulus, but in view of the unpredictable occurrence of fatal arsenical encephalopathy in a small proportion of patients receiving Mel W, suramin remains the preferred macrofilaricide in the treatment of onchocerciasis. It is therefore important to determine the suramin dosage schedules that will produce parasitological cures with minimal concomitant risks of toxic reactions. The criteria by which cures should be judged must also be appreciated.Courses of different duration and employing doses of 1.0 g, 0.5 g and 0.25 g suramin have been investigated for their action on adult worms and microfilariae, for the toxic reactions which they produce on the kidney, and for the level of suramin reached in the serum during treatment. A course of 5 or 6 weekly doses of 1.0 g has been shown to be the optimum for adults.It is sometimes stated without good foundation that different brands of suramin differ in their purity and chemical composition, thus producing inconsistent effects on the parasites and grave risks of toxic manifestations. The three main brands in current use in Africa (Antrypol, Moranyl and Naganol) have therefore been compared in a group of patients, using the same optimum dosage schedule for each preparation and recording the action of the various brands on the parasites, as well as their comparative toxicities and the serum suramin levels achieved. No significant differences between the brands were recorded.

Clinical Trials as Topic↗
PubMed1968

Experiments on the chemoprophylaxis of Onchocerca volvulus infection.

Previous work on monkeys and on human volunteers led to the development of a schedule of diethylcarbamazine dosage suitable for the chemoprophylaxis of loiasis. In several parts of Africa where this chemoprophylaxis is practised against Loa loa, infections with Onchocerca volvulus are also common. Attempts were therefore made to determine whether diethylcarbamazine has any prophylactic action against the latter parasite by making use of chimpanzees exposed to experimental infections, and also by using biopsy techniques to study the fate of infective larvae inoculated into volunteers.Both experimental methods proved more difficult to apply in O. volvulus infections than had been the case with L. loa, but evidence was obtained that diethylcarbamazine is not an effective chemoprophylactic for O. volvulus.Further experiments were then carried out with suramin and with melarsonyl potassium (Mel W). Although both gave some evidence of partial prophylactic activity, their use for this purpose is at present neither practical nor safe.

Animals↗
PubMed1977

Worm burden of onchocerca volvulus in a hyperendemic village of the rain-forest in West Africa.

During a study on the effects of nodulectomy on onchocerciasis in a village in the Liberian rain-forest 400 Onchocerca-nodules were excised from 34 inhabitants. The analysis of the nodules revealed a worm burden of 718 complete live male and 1062 complete live female worms as well as 9 male and 117 female worms which were mostly calcified and degenerating. 73% of the live females contained developing embryos or released microfilariae, the other females either contained eggs or showed empty uteri. Some observations were made on degenerating worms. The male:female sex ratio was 1:1.5 and possible reasons for this are discussed.

Adolescent↗
PubMed1981

Selective recovery of living microfilariae from Onchocerca volvulus nodules. Determination of optimal conditions for their culture in vitro for excretory/secretory products.

A method for the selective recovery of living microfilariae from Onchocerca volvulus nodules is described. The microfilariae migrate through solidified agar gel into overlayering Hanks balanced salt solution (HBSS). The highest recovery rates of the worms were obtained with 0.3 and 0.4% agar. Optimal conditions for in vitro cultures of the larvae in HBSS were established; pH range 7.0-7.5, glucose concentration 2-5 mg/ml for long term cultures, osmolality 200-309 mOsmoles/l, temperature 4 degrees C for prolonged cultures and 24-28 degrees C for overall best yield of excretory/secretory products (ESP). Subculturing of the larvae reduced contamination of ESP with human serum protein to minimal amounts after 9 recultures done within 96 h.

Animals↗
PubMed1981

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.

Animals↗
PubMed1982

Detection of IgE antibodies in onchocerciasis. Possibility of using allergens from Dipetalonema viteae extracts that cross-react with allergenic determinants in crude extracts of Onchocerca volvulus.

The present study reports the presence of Onchocerca volvulus specific IgE in the sera obtained from onchocerciasis patients. About 70% of onchocerciasis patients showed a raised level of O. volvulus specific IgE compared to patients infected either with other human filarids (Loa loa, Wuchereria bancrofti, Brugia malayi) or with other helminths (Schistosoma mansoni, Ascaris lumbricoides, Fasciola hepatica). The O. volvulus specific IgE level was significantly higher in patients exhibiting 'gale filarienne' than in microfilaremic patients or in endemic controls. The total IgE level was significantly raised in the serum samples of all groups of subjects from endemic areas compared to European controls. There was no significant increase in the level of IgE in the onchocerciasis sera when O. volvulus antigen was replaced by the antigens from various helminths in the present assay system (radioallergosorbent test). However, there was a clear evidence of the presence of cross-reacting allergens in the crude extracts from adults of O. volvulus and Dipetalonema viteae (a rodent filarial parasite) because there was a significant reduction in IgE level in onchocerciasis sera following absorption with either O. volvulus or D. viteae sorbents. Moreover, the IgE antibodies in onchocerciasis patients sera recognized the allergens which were present in the somatic extracts of O. volvulus and D. viteae as revealed by radiolabelled anti-IgE.

Allergens↗
PubMed1982

The identification of some Onchocerca spp of cattle by isoenzyme analysis.

The isoenzyme patterns of LDH, MDH, PEP, PGM, MPI, GPI and GAPDH were compared for 3 species of cattle parasites Onchocerca gutturosa, O. gibsoni and O. lienalis from England, Australia and Mexico. LDH and GAPDH had identical patterns in each species but the other enzymes clearly distinguished between the species. PGM was the same in O. lienalis and O. gibsoni, but separated them from O. gutturosa. MPI and GPI had similar isoenzymes in O. gutturosa and O. gibsoni, but distinguished them from O. lienalis. Different isoenzyme patterns for all 3 species were obtained with MDH and PEP. The potential use of these techniques towards a better understanding of the epidemiology of human onchocerciasis is discussed.

Animals↗
PubMed1982

Morphological characteristics and isoenzyme patterns of an Onchocerca sp. found in cattle in Southern Mexico.

An Onchocerca sp. was isolated from the nuchal ligaments of 78 out of 79 naturally infected cattle in Southern Mexico. Infections were moderate to heavy, averaging 20-30 parasites per nuchal ligament. Microfilariae were concentrated in the skin of the head and neck, although small numbers were found in the skin of the withers, brisket and medial abdomen. High numbers of microfilariae were found in the skin around the eye, but not in the ocular tissues themselves a situation dissimilar to that found in human onchocerciasis. Morphologically, adult females, males and microfilariae were identified as being O. gutturosa. Analysis of the isoenzyme patterns of LDH, MDH, PGM, GPI, PGD and ES of adult female worms, showed the Mexican parasite to be the same as O. gutturosa from England and Australia.

Animals↗
PubMed1983

Induction of non-specific human suppressor cells in vitro by defined Onchocerca volvulus antigens.

In the present study the activity of Onchocerca volvulus total antigens (OVA) on the proliferative response of human lymphocytes from healthy donors was investigated. Normal human lymphocytes were cultured for 72 h with polyclonal activators, phytohaemagglutinin (PHA) or pokeweed mitogen (PWM), together with OVA, and thymidine uptake was measured. The proliferation of normal lymphocytes was markedly inhibited by the parasite antigens. The inhibition was not attributable to a cytotoxic effect of OVA, since at least 80% viable cells were recovered at the end of cell cultures. The inhibition was not abrogated by removal of the adherent cell population. The passage of OVA through immunosorbent column containing human antibodies to O. volvulus significantly reduced the suppressive activity of OVA. The in vitro response to mitogens (PHA, PWM) of normal human lymphocytes was suppressed by co-culture with allogeneic or syngeneic lymphocytes, which had previously been exposed for 72 h to OVA. The suppression was not abrogated by the irradiation of mononuclear cells before the exposure to OVA. A significant reduction of the suppression was however observed when OVA pre-treated cells were T cell depleted by centrifugation of E rosettes. Thus, parasite antigens, which are recognized by antibodies in infected human sera may participate in the modulation of the cellular immune response during O. volvulus infection by inducing suppressor cells. This suppression could in addition contribute to the survival of the parasite in its host.

Animals↗
PubMed1978

Ultrastructural characterization of the cuticle of Onchocerca volvulus microfilaria.

Previous ultrastructural studies on various microfilariae have suggested the presence of a trilaminar membrane at the parasite cuticle. In the present report, the formation of the cuticle of Onchocerca volvulus microfilariae was studied at different developmental stages by transmission electron microscopy of samples obtained from subcutaneous nodules of untreated patients. The cuticle is formed by the layering of surface coat fibrillar components of the hypodermal cells. No plasma membrane is found at the cuticle of the microfilaria. The use of freeze-fracture replication confirmed the absence of a classic plasma membrane at the parasite cuticle. Our results suggest that immunogenic determinants of the microfilaria cellular elements are hidden from the exterior by the acellular cuticle. This may explain the lack of cellular reaction usually found around living O. volvulus microfilariae in the dermis of onchocerciasis patients.

Animals↗
PubMed1983

Aggregation of equine platelets by Onchocerca cervicalis collagen.

The arachidonic acid pathway plays an important role in many inflammatory reactions. Current evidence suggests that platelets can play a central part in host inflammation. Since microfilariae are mobilized into the bloodstream following diethylcarbamazine (DEC) treatment, we have studied the effects of Onchocerca cervicalis cuticle preparations on equine platelet aggregation. The authors have found that O cervicalis cuticular preparations can induce platelet aggregation in vitro. Furthermore, this activity was abrogated by treatment with collagenase and not hyaluronidase, elastase, or alpha-chymotrypsin. When this evidence is viewed collectively with the evidence for in vivo parasite cuticular damage following DEC treatment, it becomes entirely plausible that the cuticular damage may indeed reveal a platelet-reactive surface, thus permitting platelet-parasite binding to occur. This binding would result in platelet aggregation and the generation and release of platelet-derived arachidonate metabolites. These metabolites may play a very critical role in the development of the described pathologic sequelae observed following DEC treatment. Field studies using cyclooxygenase and lipoxygenase inhibitors might therefore be very efficacious in decreasing the frequency of side effects due to DEC or other potentially effective drug regimens.

Adenine↗
PubMed1984

Immunity to onchocerca lienalis microfilariae in mice. I. Resistance induced by the homologous parasite.

The model of Onchocerca lienalis microfilariae injected into inbred CBA/HT6T6 mice has been examined for its value to study immunity to the skin-dwelling microfilariae in onchocerciasis. Mice injected with living microfilariae during primary or secondary infections exhibited a high level of resistance to challenge relative to normal controls (91-98% reduction in recoveries). The survival of microfilariae during a primary infection was significantly prolonged in T-cell deprived animals compared with immunologically intact mice. Serum and spleen cells transferred from donors 90 days after infection conferred significant protection in syngeneic recipients to challenge with microfilariae (59% reduction in recoveries compared with controls). Boostered injections with freeze-killed or fragmented microfilariae reduced parasite recoveries after challenge by 39-78%: none of 5 adjuvant preparations enhanced the protective effect. Mice exposed to living infective larvae or adult males of O. lienalis also exhibited lowered recoveries of microfilariae following a challenge infection. It is concluded that the mouse model offers potential for immunological studies on the microfilariae in onchocerciasis, which have hitherto been limited because of the lack of suitable laboratory hosts.

Adjuvants, Immunologic↗
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↗
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↗
PubMed1984

The effects of diethylcarbamazine, mebendazole and levamisole on Onchocerca volvulus in vivo and in vitro.

The effects of mebendazole, levamisole, diethylcarbamazine citrate (DEC-C), and the combination of mebendazole and levamisole, on adult Onchocerca volvulus and on the in utero development of microfilariae was studied in nodules excised from patients in Southern Mexico. Adult worms isolated from patients treated with mebendazole showed a reduction in mobility and contained 40 times fewer developing microfilarial forms than did worms from untreated patients. Most of the developmental forms found in adult worms from mebendazole-treated patients were either oocytes or early morulae, with more mature forms being scarce or morphologically abnormal. Treatment with levamisole had a similar effect on embryogenesis, however it was much less marked. The number and distribution of developing forms in worms from patients treated with DEC-C was similar to that found in the control groups. The effect of the same three drugs on microfilariae in vitro was also tested. DEC-C at concentrations of 0.5 microgram - 2000 micrograms/ml did not have any obvious effect on motility or morphology of the worms provided the pH was maintained at physiological levels; levamisole also had no effect in vitro. Mebendazole induced the death of microfilariae when used at concentrations greater than 100 micrograms/ml, however these levels are greater than those found in the blood of patients under treatment. The optimal conditions for short-term maintenance of O. volvulus microfilariae in culture, are discussed. It is apparent from these studies that mebendazole has an effect on the maturation of microfilariae in utero and may also, at high concentrations, have a direct effect on mature microfilariae.

Animals↗