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PubMed1985

Preliminary data on the genetic differentiation of Onchocerca volvulus in Africa (Nematoda: Filarioidea).

Data are reported on the genetic structure of three Onchocerca volvulus populations, respectively from Mali (savanna), Ivory Coast (forest), and Zaire (forest gallery in savanna). Electrophoretic analysis, carried out on 25 gene-enzyme systems, has shown a remarkable genetic heterogeneity existing within O.volvulus. Zaire and West Africa populations appear chiefly differentiated at Mdh-1 and 6Pgdh loci, their average Nei's genetic distance being 0.11. In West Africa Nei's D found between the savanna and forest samples is 0.04. The savanna population from Zaire is more similar to the savanna one from Mali (D = 0.09) than to the forest one from Ivory Coast (D = 0.13). This appears mainly due to the loci Ldh and Hbdh (possibly linked), some alleles of which seem to be selected for in forest populations (Ldh110, Hbdh108), while others in the savanna ones (Ldh100, Hbdh100). The hypothesis that the discrepant epidemiological patterns of human onchocerciasis are related to intrinsic differences in the parasite seems supported by the obtained data. The differences in allele frequencies found at the reported loci appear strong enough to allow biochemical identification of O. volvulus populations from different geographic regions and different habitats.

Alleles↗
PubMed1986

Detection of protein kinase substrates in extracts of Onchocerca volvulus.

Extracts of Onchocerca volvulus were phosphorylated in the presence of (gamma 32P)ATP and Mg2+ by endogenous protein kinase activity and exogenous rabbit muscle catalytic sub-unit of the adenosine 3'5' monophosphate dependent protein kinase (E.C. 2.7.1.37). Sodium dodecylsulfate polyacrylamide gel electrophoretic analysis of the 32P-labelled extracts revealed at least seven (32P)-phosphoproteins with apparent Mr of 92,000; 86,000; 40,000; 27,000; 26,000; 23,000 and 17,000. The phosphorylation of the components with apparent Mr of 23,000 and 17,000 was catalysed by both endogenous and exogenous protein kinases, whereas the other components required exogenous protein kinase for their phosphorylation. The endogenous protein kinase activity was inhibited by suramin and the heat-stable protein inhibitor of the adenosine 3'5' monophosphate dependent protein kinase. The (32P)phosphoproteins identified in this investigation are probably candidate regulatory molecules in O. volvulus; though their physiological functions remain to be determined.

Animals↗
PubMed1987

Population dynamics of Onchocerca volvulus after 7 to 8 years of vector control in West Africa.

In an attempt to describe the changing population dynamics of Onchocerca volvulus during a period of vector control, nodulectomies were undertaken in 256 patients from ten villages in the Onchocerciasis Control Programme (OCP) and in 74 patients from two villages in an area with ongoing transmission. A total of 1198 nodules were excised and 4350 adult worms were isolated and examined for viability and productivity. In the OCP villages, the worm population is ageing and dying without replacement by new generations of parasites and various findings signal a breakdown of the worm population after about 12 years interruption of transmission. The sexual activity of the worms was significantly reduced. A Productivity Index was developed to measure the microfilariae production at the nodule level. The reduction in this index for the OCP villages correlates closely with the decline over the control period in the community microfilarial loads in the skin. The results show that it is not only the longevity of the parasite which will determine the duration of vector control, but that the reduced productivity of the ageing parasite population is of equal importance.

Africa, Western↗
PubMed1988

Moulting and exsheathment of the infective larvae of Onchocerca lienalis (Filarioidea) in vitro.

Fourth-stage larvae (L4) of the cattle filarial parasite Onchocerca lienalis were produced from third stage forms (L3) and maintained in vitro using a variety of culture media and feeder cell layers. Up to 50% of the L3 larvae moulted to the fourth stage in the presence of two jird (Meriones unguiculatus) cell lines, a monkey kidney cell line and also with Vero cells. Moulting took place 2-5 days after of the initiation of the cultures: These L4 parasites could be maintained in a healthy condition for up to 88 days, although no significant increases in size of these larvae were observed during the culture period. The medium giving the most promising results, both in terms of moulting and survival, was 199 supplemented with heat inactivated foetal bovine serum (20%) and glucose (2 mg/ml). The feeder layer cells could be replaced by fresh jird red blood cells. Moulting of parasites occurred in medium alone but the viability was reduced under these conditions.

Animals↗
PubMed1989

An in vitro radiolabel uptake viability assay for Onchocerca microfilariae.

A radiolabel uptake viability assay for Onchocerca cervicalis using [3H]2-deoxy-D-glucose in Hanks' balanced salt solution, pH 7.5, at 30 C is described and compared to the traditional visual motility assay. A correlation of r = 0.92 between the assays was found, with the radiolabel uptake method apparently a more sensitive indicator of microfilarial viability.

Animals↗
PubMed1986

Phosphoenolpyruvate carboxykinase from Onchocerca volvulus and O. gibsoni.

Phosphoenolpyruvate carboxykinase, a rate-limiting enzyme at the branchpoint of phosphoenolpyruvate (PEP), was demonstrated in Onchocerca volvulus and O. gibsoni. The activity of PEP-carboxykinase from both filarial worms depends absolutely on the presence of divalent cations; in addition to Mg2+ the enzyme activity was strongly activated by Mn2+. The Michaelis constants for PEP, GDP and KHCO3 of the PEP-carboxykinase from O. volvulus were determined to be 0.16 mM, 0.15 mM and 20 mM, respectively; those of the enzyme from O. gibsoni were 0.16 mM, 0.13 mM and 12 mM. Quinolinate was found to be a potent inhibitor of the enzyme from both filarial worms. The inhibition constants were determined to be 11 microM and 15 microM for the enzyme from O. volvulus and O. gibsoni. It is suggested that the activity of PEP-carboxykinase, the initial enzyme of the alternate route from PEP to succinate, may be regulated by ATP-levels. The inhibition constants for ATP were determined to be 0.26 mM and 0.13 mM for the enzyme from O. volvulus and O. gibsoni.

Adenosine Triphosphate↗
PubMed1985

The fine structure of adult Onchocerca volvulus recovered by collagenase digestion.

Very young, middle-aged and old macrofilariae of Onchocerca volvulus were isolated alive or intact from onchocercomata using the collagenase digestion technique and studied by transmission electron microscopy. The body wall and the internal organs showed a well preserved morphology and no differences were detected compared with worms which had not been in contact with collagenase solution.

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↗
PubMed1977

Preliminary studies on the histochemical differentiation of strains of Onchocerca volvulus microfilariae in Togo.

Skin snips were taken from 75 people living in four villages of northern Togo. The 7824 microfilariae that emerged were examined by staining for the presence of acid phosphatase. Four distinct patterns of enzyme staining were observed, and descriptions of the stained microfilariae are given. The study confirms the view that a number of biological strains or variants of Onchocerca volvulus coexist in West Africa, and suggestions are made for further research that could result in the practical application of these observations in onchocerciasis control programmes.

Animals↗
PubMed1988

Emergence of Onchocerca volvulus microfilariae from skin snips before and after treatment of patients with ivermectin.

As ivermectin is supposed to have a paralyzing effect, the emergence of Onchocerca volvulus microfilariae from skin snips before and after treatment was examined. Before treatment 79.8% of the microfilariae emerged from the skin snips. Three days after treatment the microfilarial skin density had dropped to 83% (placebo), 28% (100 microgram/kg ivermectin), 19% (150 micrograms/kg) and 14% (200 micrograms/kg) of the pretreatment values but the microfilarial emergence was not altered distinctly. The majority of the remaining microfilariae were mobile and able to leave the biopsy. Only a slight, but significant, dose-dependent trend of reduced emergence could be shown. From our results no direct support is derived for the hypothesis that O. volvulus microfilariae are paralyzed or immobilized by ivermectin.

Analysis of Variance↗
PubMed1988

Experimental ocular onchocerciasis in cynomolgus monkeys. II. Chorioretinitis elicited by intravitreal Onchocerca lienalis microfilariae.

Chorioretinitis due to onchocerciasis is a major cause of blindness, and the pathogenesis is poorly understood. We have developed an experimental model for onchocercal chorioretinitis using cynomolgus monkeys (Macaca fascicularis). Two normal monkeys and two monkeys which had received prior sensitization with subcutaneous injections of live Onchocerca lienalis microfilariae were given intravitreal injections of either 0, 10, 50 or 500 live microfilariae. Posterior segment changes included disc edema, venous engorgement, retinal vasculitis, intraretinal hemorrhage, and progressive retinal pigment epithelial (RPE) disturbances. Histopathological findings included perivascular infiltrates with eosinophils, eosinophilic choroiditis, and RPE hypertrophy, hyperplasia and loss of pigment. Microfilariae in the retina had no surrounding inflammation but were found adjacent to areas of RPE alterations. Overall the inflammatory reaction in the two unsensitized monkeys was more severe than that seen in the sensitized monkeys. The retinal appearance of the monkeys resembled that found in human onchocerciasis, and this model appears to be a promising one for future investigations.

Animals↗
PubMed1988

Unique recognition of a low molecular weight Onchocerca volvulus antigen by IgG3 antibodies in chronic hyper-reactive oncho-dermatitis (Sowda).

Individual human Ig class responses to Onchocerca volvulus antigens have been evaluated by Western blotting using sera from cases of generalized onchocerciasis and chronic hyper-reactive onchocerciasis (Sowda). in all cases except IgG3 the patterns of recognition by human antibody classes were similar in Sowda and generalized onchocerciasis. Weak or undetectable responses were seen with IgG1, IgG2 and IgM. The total profiles of antigens recognized by the other Ig classes were different, although in some cases certain bands were commonly identified. The result with IgG3, however, was striking. Here, two major antigens (9 kD and 72kD) were recognized by IgG3 antibodies in Sowda sera but not generalized onchocerciasis sera. Furthermore, these two antigens were not recognised by any other Ig class, either in generalized or Sowda onchocerciasis, nor were they detected by antibodies of any class present in a collection of sera representative of other nematode infections. This difference in the IgG3 response was so pronounced that Sowda sera could be distinguished from generalized onchocerciasis sera by an IgG3-specific ELISA assay with a PBS parasite extract as the antigen. Thus, a correlation has been established between one particular clinical condition of onchocerciasis (Sowda) and a serological response, defined in terms of both the parasite antigens and an immunoglobulin class restricted antibody response.

Animals↗
PubMed1988

Proposed form to evaluate some histological aspects of macrofilarial morphology, its age dependent alterations and drug related changes in nodules of Onchocerca volvulus and O. gibsoni.

Histological assessment of drug-related pathomorphological changes in macrofilariae of the two nodule forming species Onchocerca gibsoni and O. volvulus is hampered by the variability of age-dependent signs of natural degeneration and the virtual inexistence of clinically established reference medicaments. A specific form was designed to record systematically all characteristics observed in the various organs, tissues, cell types and developmental stages inside normal and drug-affected macrofilariae. Its tabular format permits the synoptic presentation of the findings in all female and male macrofilariae from a particular treated or untreated individual. The proposed form enumerates thirty eight morphological characteristics subdivided into the six subunits cuticle, hypodermis, longitudinal muscle, genital tract, pseudocoelomic cavity and intestinal tract (gut). The normal morphology of sexually mature and productive male and female O. volvulus is described and illustrated by photomicrographs. The cuticle of the male is wrinkled and forms regular striations strictly transverse to the body's longitudinal axis. The longitudinal muscles are well developed and cover almost 100% of the circumference at the anterior and posterior end. Towards the mid-body region the muscular portion diminishes to 50-60% and is replaced there by the very voluminous lateral hypodermic chords. The male genital tract originates with its distal end of the testis close to the anterior end and runs through almost the entire body very close to the posterior end. Spermatogenesis is clearly synchronized and the various development stages are arranged in strict sequential order inside the genital tract. Each type of gonosomal cell may thus serve as a marker of the respective region of the body. The same applies for females where the paired genital tubes run through almost the entire length of the body until the two uteri unite to form a short unpaired vagina. Each of the two reproductive tracts originates at different levels in the posterior part of the female. Because of this, two distinct stages of the sexual products are normally present in sections of the two uteri in the various parts of the female macrofilariae. Towards the anterior end identical stages of embryos may be found. The wide uteri narrow shortly before the vaginal junction to less than 40 microns in the very anterior part of the body. This part is characterized by the voluminous lateral chords of hypodermis and the well developed longitudinal muscles covering greater than 50% of the circumference. Drug-related changes come to light first in this part of the female's body.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1988

Vascular perfusion of Onchocerca volvulus nodules.

Perfusion of the vascular bed was achieved in 24 freshly excised nodules of Onchocerca volvulus varying from 0.5 to 2 cm in diameter. India Ink, Microfil polymer, or acrylate perfusates were passed through the vascular supply via cannulation of superficial capsular vessels. After clearing in glycerol or methyl salicylate, or KOH corrosion in the case of the acrylate, nodules were examined microscopically. Small nodules had an extensive blood supply, diffusely distributed throughout the nodule matrix, and in close association with the coils of the worms. In bigger nodules the central area appeared more dense, and intense vascularization appeared to be more peripheral; in the largest nodules the central core was not well vascularized, but a band of heavy vascularization was seen at the margin of the core, fed by superficial vessels and in close contact with worm coils. Very fine branches of the vascular tree were perfused by all three contrast media, but histologically there was evidence of incomplete filling of the smallest vessels. However, there was no extravasation of per-fusates around parasites, even where the approximation between between vessels and parasite surfaces was close. The possibility is considered that O. volvulus may control blood vessel proliferation by release of angiogenesis factors, analogous to rapidly growing solid tumors.

Animals↗
PubMed1988

Investigations of enzymatically isolated male Onchocerca volvulus: qualitative and quantitative aspects of spermatogenesis.

A simple technique for the identification of the different spermatogenetic stages of Onchocerca volvulus is described. It is practicable for large-scale examinations in the field. The reproductivity of male worm populations can be assessed and directly correlated with the corresponding female worm populations. The activity of male filariae decreases with the increasing age of the worm population. Only few male worms transmit spermatozoa during mating, but spermatogenesis is continous also in very old male individuals. These old male filariae store high numbers of spermatozoa and cease their migration between the nodules.

Aging↗
PubMed1988

In vitro maintenance of Onchocerca volvulus for harvest of excretory and secretory products.

Recommendations are given for maintenance of female Onchocerca volvulus; conditions as standardized here for drug screening and harvest of secreted-excreted products resulted in survival of worms in culture medium for up to four and six weeks in absence and presence of serum, respectively. As parameter for viability of worms lactate excretion was determined beside control of motility.

Animals↗
PubMed1988

The fine structure of male and female Onchocerca gibsoni.

The fine structures of the midbody regions of male and female Onchocerca gibsoni are described. The epicuticle is wrinkled in the male, and forms long protuberances in the female worm. The lateral chords of the female specimens contain many bacteria and dense bodies. In both sexes the intestinal cells are filled with concentric spherules and the intestinal lumen is extremely reduced. The fine structures of O. gibsoni are very similar to those of O. volvulus.

Animals↗
PubMed1988

Reproduction in female Onchocerca gibsoni.

The embryonic stages in the uteri of adult female Onchocerca gibsoni were studied to determine the reproductive potential of this worm. It was concluded that female O. gibsoni undergo cyclic phases of reproduction, each lasting about 14.5 weeks. Once insemination occurred and a cycle commenced, the rate of embryonic development was reasonably constant irrespective of the age of the worm. Mature young females had fewer embryos than older females but both contained a similar proportion (about 19%) of abnormal embryos. In comparison to the older female worms, a greater proportion of mature young females were in the early phase of a reproductive cycle and fewer were actively reproducing. This difference was considered to be due to a greater access of males to older than to recently mature females. There were marked similarities between these observations with O. gibsoni and those reported for O. volvulus, providing further evidence of the usefulness of O. gibsoni as a model for O. volvulus.

Animals↗