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PubMed1990

The use of cell-conditioned medium for the in vitro culture of Onchocerca spp. larvae (Nematoda: Filarioidea).

The effects of co-culture with monkey kidney cells (LLCMK2), cell-conditioned medium and decreased atmospheric oxygen on the in vitro molting and viability of infective stage larvae (L3) of Onchocerca lienalis and O. volvulus were examined. O. lienalis L3 were cultured in an RPMI 1640-based medium in the presence of an LLCMK2 cell monolayer or in medium which had been conditioned for three days by cells. In paired experiments cell conditioned medium alone in 95% air/5% CO2 produced molting levels of 54 +/- 14% which increased to 67 +/- 20% in treatments cultured under decreased oxygen; this value equalled the level of molting of worms cocultured with LLCMK2 cells. Worm survival in the three environments was similar. In seven additional experiments using O. lienalis (n = 186), overall levels of 74 +/- 12 percent molting and 75 +/- 7% viability on days 21-33 were obtained. Worms increased in length from 503 +/- 50 mu as L3 to 638 +/- 74 mu as L4 on day 21 (p = 0.0001, n = 42-44). Ultrastructural comparison of an in vitro derived L4, (39 days in culture) vs a vector-derived L3 revealed fewer annulations and decreased osmiophilia on the epicuticle of the L4 while the hypodermis showed increased morphogenetic definition. O. volvulus molted at an average rate of 74% (n = 40) with a mean viability on day 28 of 95%. L3 increased in length from a mean of 635 +/- 50 mu to 775 +/- 45 mu as L4 on day 28 (p = 0.0001). Larvae of both species were cultured under these conditions for periods of time exceeding 100 days.

Animals↗
PubMed1990

Differential length of Onchocerca volvulus infective larvae from the Cameroon rain forest and savanna.

Infective larvae from the savanna strain of Onchocerca volvulus are significantly longer (662.6 +/- 66.8 microns; n = 99) than those from the forest strain (624.2 +/- 62.0 microns; n = 236). The length of the infective larvae is not influenced by the size, species or age post infectionem (pi) of the Simulium damnosum s. l. vectors nor by their localization in the fly's head, thorax or abdomen, the worm load per fly or the thoracic volume per larva. However, the lengths of infective larvae within one individual fly have a conspiciously low variance.

Analysis of Variance↗
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↗
PubMed1990

Novel Onchocerca volvulus filaricides from Carapa procera, Polyalthia suaveolens and Pachypodanthium staudtii.

Suitable drugs for the elimination of adult Onchocerca volvulus are still needed since ivermectin, the new microfilaricide, appears to be ineffective against this parasite stage. Herein we report on the identification of filaricides in three medicinal plants. The compounds carapolide A, mexicanolide-methylangolensate mixture from Carapa procera and oliverine from Polyalthia suaveolens or Pachypodanthium staudtii when tested at 10-100 micrograms.ml-1 were found to exhibit considerable microfilaricidal activity after 24 hours of incubation. Oliverine was found to be filaricidal when tested against adult female worms at 100 micrograms.ml-1. Preliminary toxicity studies in mice showed carapolide A and the mexicanolide-methylangolensate mixture to be relatively non-toxic, whereas, oliverine had a minimal lethal dose of 8 mg.Kg-1 of body weight. Based on the above results further pharmacologic studies are recommended to determine, the potential application of the active compounds on the treatment of onchocerciasis.

Animals↗
PubMed1986

Antigenic similarity of Onchocerca volvulus to other helminths examined by monoclonal antibodies against O. volvulus.

Antigens in Onchocerca volvulus were characterized using anti-O. vovulus monoclonal antibodies (McAb). A total of 17 McAb which were not species-specific to O. volvulus was examined for the antigenic similarity of O. volvulus with 12 parasitic helminths using indirect immunofluorescence. The range of reactivity of these McAb was wider in nematodes in contrast to species of cestoda or trematoda. The antigenic similarity within helminths examined was generally in agreement with their taxonomic relationships but was not shown in the lower taxonomic hierarchy. The helminth which holds the antigens most in common to O. volvulus was not the filarial parasites but Ascaris suum, an intestinal nematoda. The least antigenic similarity to O. volvulus was found in Schistosoma mansoni and S. japonicum, trematoda, among helminths examined. On the other hand, the existence of a common antigen shared with all the 13 helminths examined was demonstrated by a IgM McAb.

Antibodies, Monoclonal↗
PubMed1989

Parasitologic survey of Onchocerca volvulus infection in the Nimiyama Chiefdom, Kono District, Sierra Leone.

Infection due to Onchocerca volvulus was investigated in the Nimiyama Chiefdom, Kono District, Sierra Leone, where Simulium damnosum s.l. is known to breed but no data on the prevalence and intensity of O. volvulus infection exists. Of the 735 individuals sampled by the skin snip method in five villages, 471 (64%) were infected. The infection rate for males was significantly (p less than 0.05) higher than that of the females. Two hundred and ninety-one (70.0%) of the 416 males skin snipped and 180 (56.0%) of the 323 of their female cohorts were infected. The intensity of infection increased with age, with the younger cohorts presenting lower microfilarial density (MFD). Palpable nodules were observed in 230 (48.4%) of individuals found positive for skin microfilariae and in 37 (5.0%) individuals found negative for skin microfilariae. It is concluded that O. volvulus infection is mesoendemic in the Nimiyama Chiefdom, with the intensity of infection increasing with age.

Age Factors↗
PubMed1989

The development of Onchocerca ochengi in surrogate temperate Simuliidae, with a note on the infective larva.

Laboratory reared nulliparous female flies of six temperate species of Simuliidae were examined for their susceptibility to infection with Onchocerca ochengi by intrathoracic injection of cryopreserved skin microfilariae obtained from cattle in Mali. Three species (S. equinum, S. ornatum and S. erythrocephalum) supported development to the infective stage, one species (S. variegatum) allowed partial development and the remaining two species (S. reptans and S. aureum) were insusceptible to infection. The most suitable surrogate vectors were S. equinum and S. ornatum which had survival rates of 44% and 49%, proportions of microfilariae developing to third stage larvae of 6.4% and 3%, and infection rates with infective larvae of 13.5% and 14% respectively. O. volvulus infective larvae, produced by intrathoracic microfilarial injection in S. ornatum, were 586-760 microns (mean 687 microns) long and were significantly shorter (p less than 0.02) than the O. ochengi infective larvae (645-880 microns, mean 756 microns). No constant differences in the posterior or anterior morphology, or in the acid phosphatase staining patterns between O. ochengi and O. volvulus, were seen. These results raise the possibility that the presence of O. ochengi in a population of infective larvae from vector flies in endemic onchocerciasis zones might be identifiable on the basis of their length alone.

Animals↗
PubMed1989

Electron microscopic studies on the effects of CGP 6140 and CGP 20376 on microfilariae and third stage larvae of Onchocerca volvulus.

Electron microscopic examination was used to assess the effects of CGP 6140 (proposed generic name: amocarcine) and CGP 20376 (proposed generic name: metobethiamide) on the fine structure of microfilariae and third stage larvae of Onchocerca volvulus after in vitro exposure to different concentrations of these compounds. The range of concentrations was selected according to the expected plasma levels that may be reached with these drugs. The microfilariae showed distinct effects on the muscle cells as myelin figures, vacuolisation and disintegration of the cytoplasm after both compounds. The third stage larvae showed the same effects on the muscle cells including changes in the mitochondria and the hypodermal tissue. In addition, the glandular esophagus demonstrated degenerative alterations of the cytoplasm and loosening of the cuticular lining after exposure to CGP 6140. CGP 20376 led to severe damage of the fibres of the nerve ring. No degenerative changes of the microtubuli were observed. Comparison of the morphological effects observed by electron microscopy with the reduction rates of motility and moulting recorded in the in vitro tests showed good correlation between these different parameters. The observed effect on the fine structure was the most sensitive parameter.

Animals↗
PubMed1989

Analysis of ecdysteroids in Onchocerca gibsoni, O. volvulus and nodule tissues.

Free ecdysteroids were detected in Onchocerca gibsoni, in tissues constituting O. volvulus and O. gibsoni nodules and in unrelated bovine tissues. Ecdysone and 20-hydroxyecdysone were identified by HPLC-RIA and GC/MS(SIM). The concentration of free ecdysteroids in the nodule tissue immediately surrounding the parasites was at least an order of magnitude higher than that detected in the worms themselves, or in adjacent nodular tissues or other bovine tissues.

Abattoirs↗
PubMed1989

Immunity to Onchocerca volvulus. Serum mediated leucocyte adherence to infective larvae in vitro.

Leucocyte adherence to infective larvae of Onchocerca volvulus in the presence of serum was evaluated using sera from four clinically distinct groups of patients with Onchocerciasis from an area hyperendemic for the disease. Significant cellular adherence to infective larvae occurred for the most part in the presence of sera obtained from subjects with either no microfilaridemia and few or no palpable nodules. These patients had, as well, the highest serum titres of specific anti-O. volvulus IgG antibodies. In contrast, sera from subjects with many palpable nodules and heavy skin infiltration with microfilariae (generalised disease) did not mediate significant adherence of leucocytes to infective larvae targets. Further, this group had the lowest serum levels of specific anti-O. volvulus antibodies of the IgG isotype. The findings are in keeping with the hypothesis that specific protective immunity may occur in O. volvulus infections.

Animals↗
PubMed1989

Studies on the activity of the Ciba Geigy compounds CGP 6140, 20376, 20309 and 21833 against third and fourth stage larvae of Onchocerca volvulus.

The effect of four new antifilarial compounds, (CGP 6140, 20376, 20309 and 21833, Ciba Geigy Ltd. Basle) on the viability of third and fourth stage larvae of Onchocerca volvulus was tested in vitro and in vivo. The motility of the larval stages and the moulting process from the third to the fourth stage were used as parameters for the drug activity in vitro. All four compounds showed good effects in vitro on the moulting rates compared to the controls after incubation at different concentrations in culture medium with serum. After an exposure for three hours to different concentrations of the compounds in medium without serum, only CGP 20376 had a clear inhibitory effect on the developing larvae. The moulting rate was reduced by this compound to 20% of the control values with 1 microgram/ml, a concentration that is comparable to plasma levels reported from chimpanzees. In both experimental situations, with serum and without serum, the motility of the developing larvae was only affected at very high concentrations of the compounds. The results obtained with fourth stage larvae were comparable. CGP 6140 and CGP 20376 were tested in vivo, using a diffusion chamber technique for the implantation of infective larvae into the peritoneum of Mastomys natalensis and assessing the survival of the implanted parasites. High doses of CGP 6140 had only unsatisfactory effects on the larvae in this model system whereas CGP 20376 had inhibited moulting and killed most larvae with a daily dose of 25 mg/kg for five consecutive days.

Animals↗
PubMed1987

Lectin binding to the developing forms of Onchocerca gutturosa microfilariae.

Uterine and skin derived forms of microfilariae (mf) and earlier developing stages of the cattle filarial nematode Onchocerca gutturosa were examined for the lectin binding properties of their external surfaces (ie. egg shell and microfilarial cuticle). Both the uterine and skin derived forms of mf failed to bind any of the lectins tested. Incubation in known microfilaricides did not promote any binding of lectins to these parasites. Peanut agglutinin (PNA) was the only lectin found to bind specifically to earlier developing stages (i.e. the shells of freshly obtained eggs). Diethylcarbamazine (DEC) did not alter the binding pattern from that observed in untreated eggs. Ivermectin eliminated PNA binding but increased Ricinus communis lectin (RCA-1) positivity. Trypsin, collagenase and protease enzymes all affected the binding; chitinase however did not have any effect. These results support the concept that the eggshells of filariae can interact with the host defence responses.

Animals↗
PubMed1988

Isolation and biochemical composition of the cuticle of Onchocerca volvulus.

A preparation of the cuticle of Onchocerca volvulus was obtained by extracting worm fragments in an series of buffers with 1.5% Triton-X-100 and 3% Sodium dodecyl sulfate (SDS). Electron micrographs of worm fragments, treated with the detergents or collagenase showed that our methods had been effective in isolating the cuticle from the other organs of the parasite. The cuticular preparation was found to contain 19 different amino acids with glycine (23.4%); proline (11.23%); hydroxyproline (10%); and glutamic acid (9.4%) being the most abundant. Hydroxylysine was present in small amounts (0.04%). Total reducing sugar was determined to be 5.3 mg per gram dry weight of the preparation. The cuticular preparation was solubilized by boiling in 2-mercaptoethanol and shown by SDS-PAGE to contain at least 10 different polypeptides in the Mr range 17,000-163,000. Five of these polypeptides with apparent Mr respectively of 33,000; 67,000; 74,000, 88,500 and 114,000 were isolated by preparative gel electrophoresis and their amino acid compositions shown to be similar to that of invertebrate collagens. We conclude that the cuticle of O. volvulus contains collagen-like proteins held together by disulfide bridges.

Amino Acids↗
PubMed1988

Histological examination of adult Onchocerca volvulus and comparison with the collagenase technique.

The methods used for the assessment of adult Onchocerca volvulus by histology are described. Based on the results of several studies, mainly in Liberia and Burkina Faso, the morphology of the adult filariae in histological sections is represented as far as it is relevant for the evaluation. Especially are described the morphological alterations due to old age of the worms, to chronic hyperreactivity of the human host (sowda) and effects of the macrofilaricidal suramin and of microfilaricidal drugs. Quantitative results are reported on untreated adult O. volvulus from various countries, the changes of the worm population during 12 years of vector control in Burkina Faso and the effects of suramin, diethylcarbamazine, metrifonate, and ivermectin. The data from the histological examinations are compared with those gained from the examination of worms isolated by the collagenase technique in the same studies.

Aging↗
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↗
PubMed1988

In vitro motility of isolated adults and segments of Onchocerca volvulus, Brugia pahangi and Acanthocheilonema viteae.

Observations were made on the spontaneous motility in vitro of entire adult worms and segments of Onchocerca volvulus, Brugia pahangi and Acanthocheilonema viteae. Segment motility was recorded in a mechano-transducer apparatus and responses of worm tissues to anthelmintics and other pharmacological agents were compared. Entire adult female O. volvulus showed varied patterns of motor activity, ranging from continuous low level whole body motion to periodic spasmodic contractions interspersed between intervals of quiescence lasting from a few to 30 seconds. The same range characterized movements of 1.5-4 cm segments cut from worms liberated from collagenase digested nodules. However, fresh segments dissected directly from surgical specimens were completely inactive due to the paralyzing effect of xylocaine, used as local anesthetic. This effect wore off in 2-5 hours in vitro, and recovered segments behaved in the same way as those from enzymatically liberated worms. Segments of B. pahangi and A. viteae also showed motor activity patterns which reflected those of whole adults. Segments of O. volvulus behaved reproducibly, whether examined in the field in endemic areas, or after transport of nodules or freed worms to Michigan from Guatemala or Sudan. Segments of all worms were unresponsive to most anthelmintics, but O. volvulus was susceptible to paralysis by CGP 6140, levamisole, pyrantel and carbachol at concentrations of ten to a thousand times lower than those required to produce any influence on B. pahangi. Segments of A. viteae more closely resembled O. volvulus in their dose responses to these drugs, although CGP 6140 was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Isolation and in vitro translation of Onchocerca volvulus mRNA.

Adult Onchocerca volvulus recovered for excised nodules by dissection or treatment with collagenase have been used as a source of RNA for in vitro translation experiments. RNA was purified using either the hot phenol/SDS procedure or the guanidine isothiocyanate protocol. Immunoprecipitation experiments performed on in vitro products demonstrate a marked heterogeneity in responses by individed human infection sera. Further immunoprecipitation experiments demonstrate cross reactivity between O. volvulus and other filarial nematodes.

Animals↗