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PubMed1987

Implantation of adult Onchocerca gutturosa into laboratory rodents.

Adult males and females of Onchocerca gutturosa were implanted into the peritoneal cavity of mice, and their survival was determined at intervals thereafter by post-mortem examination. Ten of 17 animals receiving males dissected free of bovine host tissues contained live parasites at necropsy up to 4 months later. Female worms digested free from connective tissue by collagenase did not survive well, even though they appeared motile and intact before implantation; only 2 were alive 10 days later in 2 of 16 recipients. When males and females still contained within connective tissue capsules were implanted they survived for up to 2 months, and microfilariae were detected transiently in the skin of 2 recipient mice. The results suggest the feasibility of maintaining adult O. gutturosa in rodents by this means, provided exposure of worms to enzymes used to free them from host tissue is avoided.

Animals↗
PubMed1989

A preliminary assessment of the feasibility of evaluating promising antifilarials in vitro against adult Onchocerca volvulus.

The suitability of motility indices and tetrazolium-based colorimetric assays for the determination of the viability of adult Onchocerca volvulus after in vitro exposure to potential macrofilaricides has been examined. Experimentation showed that both techniques could be applied to adult O. volvulus, although the variability between individual worms necessitated the use of large experimental groups. The potential of using cut anterior tips of female O. volvulus for screening was also investigated. These were shown to give reasonably consistent motility indices, and drug effects were discernible even after 72 h in vitro culture. Application of these viability criteria to studies on the short-term in vitro survival of intact male and female O. volvulus incubated in Eagles MEM plus serum, under 5% CO2 in air, showed this medium to be suboptimal with a greater than 50% loss of worm viability within 144 h of nodulectomy. Males isolated by the collagenase technique were shown to be significantly less viable than dissected males, by both motility indices and tetrazolium reduction. The results highlight the need to use either dissected males, or in the case of females, the need to minimize exposure to collagenase solution. A possible mechanism for selecting a more uniformly viable female worm population is discussed. Examination of the in vitro effects of CGP 20376 using these viability criteria/assay systems showed some delayed suppression of worm motility, but after 120 h in vitro CGP 20376 was not macrofilaricidal against male or female O. volvulus. Male worms were also implanted subcutaneously into gerbils.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Comparison of glycolysis and glutaminolysis in Onchocerca volvulus and Brugia pahangi by 13C nuclear magnetic resonance spectroscopy.

Comparison of glycolysis in Brugia pahangi and Onchocerca volvulus by 13C nuclear magnetic resonance (NMR) spectroscopy showed that the former organism is predominantly a lactate fermenter and the latter resembles more closely the metabolism of a mixed acid fermenter producing lactate, succinate, acetate, ethanol, formate and carbon dioxide. Both organisms synthesize glycogen as a storage carbohydrate. Glutaminolysis in both organisms proceeds by the delta-amino-butyrate shunt to produce succinate which is then further metabolized to acetate and carbon dioxide as end-products.

Animals↗
PubMed1992

Age-related differences in parasitosis may indicate acquired immunity against microfilariae in cattle naturally infected with Onchocerca ochengi.

Onchocerca ochengi is a common parasite of cattle in savanna areas of West Africa and its adult stages inhabit intradermal nodules. We have exploited the accessibility of the macrofilariae to examine quantitative and qualitative differences in both the macro- and microfilariae (mff) in relation to age in naturally infected cattle. Autochthonous cattle at a site on the Adamawa plateau, North Cameroon, situated near a perennial Simulium damnosum s.l. breeding site, were examined in three age groups (1.5-2.5, n = 34; 3-5, n = 39 and greater than or equal to 8 years old, n = 21). Skin mff densities were assessed from ventral skin biopsies, total body counts of nodules were done by palpation and 1-4 nodules were excised from positive animals, collagenase digested and the numbers and condition of macrofilariae determined. Embryogenesis profiles (embryograms) were done on a representative number of female worms in each age group. The overall prevalence of infection in the study population was 71% for mff and 85% for nodules. Nodule prevalence increased significantly from the 1.5-2.5 to 3-5 years old groups, but no other inter-age prevalences were significantly different. While the mean number of nodules per animal increased with age, geometric means (gm) 3.7, 15.5 and 23.1, the skin mff density maximized in the middle age group and decreased significantly in the old animals (gm 15.8, 44.7 and 11.4 mff/g, difference between latter two parameters significant, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1986

Acquired resistance to Onchocerca lienalis infections in Simulium ornatum Meigen and Simulium lineatum Meigen following passive transfer of haemolymph from previously infected simuliids (Diptera, Simuliidae).

This study describes experiments which demonstrate the presence of a parasite-induced or derived factor in the haemocoel of Onchocerca lienalis-infected simuliids. Haemolymph, when transferred from previously infected donor flies to previously untreated recipient flies, using fine glass needles, confers protection against a subsequent challenge to the recipients. In three trials using Simulium ornatum sl. or S. lineatum, significant levels of protection in 'immunized' compared to 'challenge control' groups were achieved (84% and 81% with S. ornatum sl. and 85% with S. lineatum). This is despite the fact that the worm burdens of the infected donor groups in the three trials were very different (means of 33.1 and 3.4 larvae/fly with S. ornatum sl. and 1.6 larvae/fly with S. lineatum). The use of sham-operated and bentonite-inoculated control groups demonstrate that this effect is not merely a non-specific reaction to trauma, but is probably parasitic in its derivation. The use of double infections separated by 4 days shows that the donor flies have themselves probably acquired resistance to O. lienalis. Frequency distributions of O. lienalis burdens in individual flies show that there is a shift from a normal to a skew distribution when comparing 'challenge control' flies with 'immunized'. This possible acquired resistance could have important implications in the transmission of forest and savanna human onchocerciasis by the Simulium damnosum complex.

Animals↗
PubMed1996

Susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to nitric oxide and hydrogen peroxide in cell-free culture and from IFN gamma-activated macrophages.

The susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to H2O2 and NO either in cell-free culture or from IFN gamma-activated macrophages was examined. In cell-free culture, O. lienalis microfilariae were highly susceptible to H2O2 induced toxicity, exhibiting rapid reductions in motility and viability. The addition of exogenous catalase abrogated H2O2-induced killing. In contrast, B. malayi microfilariae were relatively resistant to H2O2, with concentrations as high as 50 microM having no effect on motility or viability. On exposure to NO, both species showed reductions in motility within 5-30 min, but longer was required to see effects on the viability of microfilariae. Parasites incubated with IFN gamma-activated macrophages also exhibited marked reductions in motility and viability. In cultures with B. malayi and activated macrophages, inhibition of these effects was achieved by the addition of either L-NMMA, to abolish NO production, or neutralizing anti-TNF alpha antibodies. Attempts to inhibit parasite killing by the addition of catalase to macrophage cultures were ineffective. The results of this study show that B. malayi and O. lienalis microfilariae have different susceptibility to H2O2, but are equally affected by exposure to NO. Moreover both species are killed by IFN gamma-activated macrophages and in the case of B. malayi, killing is dependent on the generation of NO via TNF alpha.

Animals↗
PubMed1996

The ultrastructure of the anterior end of male Onchocerca volvulus: papillae, amphids, nerve ring and first indication of an excretory system in the adult filarial worm.

A detailed morphological investigation of the anterior sensory organs, the nerve ring and a glomerulus-like structure in male Onchocerca volvulus was performed by means of electron microscopy. The 8 head papillae are arranged in the common 4 + 4 pattern of most filarial worms in circles around the mouth opening. The amphidial openings are found between the circles of inner and outer papillae on both sides of the mouth. Inside, several additional nerve axons are seen in the tissue of the anterior tip not related to one of the identified papillar structures. The inner and outer papillae exhibit a remarkably different fine structure, and are part of a complex system of at least 2 different receptor cell types at the anterior tip of the worm. The amphidial channel contains 8 modified cilia; accessory axons are associated with the cytoplasm of the sheath cell. The anterior nerve ring of male worms is located about 150 micrometers posterior from the outermost tip of the head region. It consists of several fibres coiled around the oesophagus. The comparison of the fine structure of the central nervous system did not show the expected morphological differences associated with the heterogeneous age distribution in the natural worm population. This was in contrast to previous findings with respect to tissues in different parts of the worm. The study also provides the first evidence that suggests the existence of an excretory organ in a filarial worm in the region of the anterior nerve ring. Paired glomerulus-like structures in the lateral chords and a canal formed by a projection of the basal zone of the cuticles were identified.

Animals↗
PubMed1996

Neutrophil granule proteins: evidence for the participation in the host reaction to skin microfilariae of Onchocerca volvulus after diethylcarbamazine administration.

The participation of neutrophil granulocytes in the cellular reaction to skin microfilariae of Onchocerca volvulus was studied by immunohistochemistry. Skin biopsies were obtained from adult Liberian and Ugandan patients with generalized onchocerciasis after exposure to topically applied diethylcarbamazine (DEC) and from untreated patients. After DEC many damaged microfilariae were observed either in dermal infiltrates or in epidermal microabscesses consisting both of neutrophils and eosinophils. Infiltrates and microabscesses contained some intact granulocytes and many neutrophils releasing myeloperoxidase, elastase, lactoferrin, defensin, lysozyme, alpha 1-antitrypsin and alpha 1-antichymotrypsin. Eosinophils discharged peroxidase and cationic proteins. Released granule proteins and remnants of disrupted granulocytes were found on the surface and in close proximity of damaged microfilariae in dermal infiltrates and epidermal microabscesses. In larger microabscesses neutrophils were predominant. These observations show that neutrophils and not only eosinophils recruit, accumulate, localize around and release their helminthotoxic granule proteins such as myeloperoxidase onto or closely around skin microfilariae of O. volvulus after topical DEC administration. The association between these processes and the damage of the microfilariae indicated that neutrophils together with eosinophils attack and damage microfilariae of O. volvulus after DEC treatment in the skin.

Animals↗
PubMed1996

Strain differentiation of Onchocerca volvulus from Uganda using DNA probes.

Polymerase chain reaction (PCR) combined with non-radioactive DNA hybridization was applied for the detection and characterization of a 150 bp tandem repeat of Onchocerca volvulus. DNA of worms from western Uganda was amplified and then probed with a digoxygenin-labelled oligonucleotide, specific for the forest form of O. volvulus and compared to samples from various African countries. Hybridization was only observed with PCR products from the forest in Liberia, south-eastern Ghana, Benin and southern Cameroon, but not with worms from Uganda or the savannah in Burkina Faso and northern Ghana. A nested PCR using primers derived form the forest form-specific DNA sequence confirmed these results. Morphometric studies revealed length differences between the microfilariae of Ugandan O. volvulus to those of West Africa, especially to those of the savannah in Burkina Faso. It is concluded that the forest/savannah classification of O. volvulus from West Africa is not suitable for Simulium neavei-transmitted O. volvulus from Uganda.

Africa, Western↗
PubMed1993

The effects of vector control on the antibody response to antigens of Onchocerca volvulus.

The effects of exposure to infective larvae on the antibody response to a cocktail of specific recombinant antigens of Onchocerca volvulus and to a worm extract were evaluated by comparing the responses of individuals from a vector controlled area with those from an area of continuing transmission by ELISA. Individuals from the vector controlled areas were found to have reduced responses to both antigen preparations. A microfilerdermic (mf-) individuals from the area of vector control exhibited significantly lower total and subclass IgG responses to the worm extract. In contrast, the responses to the cocktail of specific recombinants were significantly reduced in individuals from the area of vector control who were still microfilerdermia positive (mf+). The distribution of IgG subclass specific responses was similar to both antigen preparations, both dominated by the IgG4 and IgG1 subclasses. IgG1 responses to the worm extract remained elevated in the vector controlled individuals but IgG4 was significantly reduced in the mf- individuals. Both subclasses reflected the total IgG response to the cocktail of recombinants and were significantly reduced in individuals from the vector controlled area, when compared to individuals from the hyperendemic area. IgG1 responses to the cocktail of recombinants are significantly lower than IgG4 in all individuals and virtually absent in individuals from the vector-controlled area. Measuring total IgG and IgG4 is more sensitive than IgG1 in detecting infection, 100 or 97% respectively, but they remain elevated in the individuals from the vector controlled areas even after 8-10 years interruption of transmission. These results have important implications for the serological monitoring of control programmes in individuals who have previously been infected.

Analysis of Variance↗
PubMed1993

Viability of Onchocerca volvulus in vitro.

The use of a selective schedule of tests to identify a viable population of isolated adult Onchocerca volvulus (Nematoda: Filarioidea) has been investigated in a large worm population. The study was initiated to develop methodology appropriate to test new candidate macrofilaricides for their in vitro activity against O. volvulus. After removal from the host the viability of isolated intact parasites was estimated by assessing the motility indices of male worms, and the colorimetric quantification of the reduction of the bioreducible tetrazolium reagent XTT and lactate output by female worms. Additionally the motility of whole females and the movement of inner organs of female worms were scored quantitatively. These response parameters were used to sort the adult worms into viability groups at the start of the in vitro culture. The adult worms were then observed for 6 days and viability was assessed regularly during the culture period. At the end of the culture period, the reduction of the water-insoluble tetrazolium reagent MTT was used to determine the formazan formed by the entire male and female worms. The response parameters used at the start of the culture proved to be highly predictive for detecting viable and non-viable adult worms. In the group of worms selected as 'viable' around 70% kept their motility and metabolic activity at a high level until the end of the culture compared to the initial level. In contrast, none of the female worms and only 13% of the male worms categorized as 'poorly viable' demonstrated a motility index or metabolic level at the end of the culture period that was comparable to that of the worms in the 'viable' groups. For female worms the lactate output correlated significantly with weight whereas no correlation was seen between MTT reduction and weight.

Animals↗
PubMed1992

Host strain, H-2 genotype and immunocompetence do not affect the survival or development of Onchocerca lienalis infective larvae implanted within micropore chambers into mice or rats.

The survival, growth and development of Onchocerca lienalis 3rd-stage (L3) larvae implanted into mice within micropore chambers has been studied with a view to developing a vaccination model for studies of protective immunity in onchocerciasis. The influence of host genetics on worm recoveries and development (growth and moulting rate) was analysed in a panel of inbred mice (CBA, BALB/c, DBA/2, SJL, 129J, C57BL/10 (B10), C3H/He and NIH), together with mice of BALB and B10 backgrounds with different major histocompatibility complex (H-2) genes (BALB/c, BALB.K, BALB.B and B10, B10.D2/n, B10.BR, B10.S). Parasite recoveries and development were similar in all mouse genotypes tested. They were unaffected by procedures designed to block or modulate phagocytic cell function with carbon or carrageenan, or to suppress inflammation by treatment with hydrocortisone acetate. A comparison of chambers sealed with membranes designed to admit (5.0 microns pore size) or exclude (0.2 microns pore size) host cells demonstrated no effect on the percentage recovery of living larvae, although dead larvae were more frequently retrieved when cells were excluded. Recoveries and rates of development of larvae implanted into immunodeficient scid mice and athymic Hooded rats were similar to those recorded in immunocompetent controls. We conclude that host genetic factors and immunocompetence are not significant determinants of survival, growth or development of O. lienalis larvae implanted within micropore chambers into naive mice. Despite its limitations, the use of this system merits further investigation as an approach to the study of protective immunity against developing larvae in onchocerciasis.

Animals↗
PubMed1993

Experimental onchocerciasis in chimpanzees: cellular responses and antigen recognition after immunization and challenge with Onchocerca volvulus infective third-stage larvae.

Immunization of chimpanzees with radiation-attenuated infective 3rd-stage larvae (L3) of Onchocerca volvulus did not induce strong protective immunity against a subsequent challenge infection; only 1 out of 4 immunized animals remained non-patent (i.e. microfilariae-negative) after challenge, and may have been protected. However, during immunization and before challenge, a broad range of adult O. volvulus-derived antigens (OvAg) and also uterus-derived OvAg were recognized by circulating antibodies; moreover, the repertory of antigens recognized increased further in subsequently patent animals after challenge, particularly in the range of M(r) 12-42 kDa. In the immunized and non-patent chimpanzee, by contrast, serological recognition of uterus-derived OvAg with M(r) 14 kDa and 105 kDa disappeared by 19 months post-challenge (p.c.). During immunization, Acanthocheilonema viteae L3 antigens of M(r) 11-12 kDa were strongly recognized only by the non-patent animal, suggesting that recognition of these antigens may have supported resistance to the subsequent challenge infection. In immunized chimpanzees, a substantial increase in the cellular reactivity to OvAg was induced; this, however, declined by 19 months p.c. to levels similar to those seen prior to immunization. At that time, 3 out of 4 immunized animals were patently infected. The effect of exogenous cytokines on in vitro-reactivity of PBMC to OvAg was examined. Addition of exogenous IL-2 alone, IFN-gamma alone, and IFN-gamma in combination with IL-2, did not augment net cellular responses to OvAg by PBMC from infected and control chimpanzees. In the presence of IL-4 alone, IL-6 alone, IL-2 with IL-4, IL-2 with IL-4 and IFN-gamma, or IL-2 with IL-4 and IL-6, the net cellular reactivity to OvAg increased significantly in patent chimpanzees and reached levels similar to non-patent animals. Thus, non-patent chimpanzees maintain high cellular reactivity to OvAg and in vitro cellular unresponsiveness to OvAg on the part of patent chimpanzees is reversible after addition of several cytokines which act individually or synergistically.

Animals↗
PubMed1995

Chemical guidance of Onchocerca lienalis microfilariae to the thorax of Simulium vittatum.

The behavioural responses of Onchocerca lienalis microfilariae (mf) to tissue factors of the surrogate black fly host, Simulium vittatum, were studied using a novel in vitro bioassay. Mf accumulated towards thoracic tissues to a density 4 times higher than towards abdominal tissues, despite the larger surface area and volume of abdominal tissues. Mf migrated toward thoracic tissues regardless of whether or not contact with thoracic tissues was possible. Therefore, mf directed themselves toward the thorax rather than arresting their movement after they make contact with thoracic tissues. Chemical cue(s) provided a principal guidance for mf to locate thoracic tissues. Mf lost their ability to differentiate thoracic and abdominal tissues following addition of thoracic attractant(s) to excised abdomens and reversed their differential response when excised thoraces were depleted of chemical cue(s). Mf did not respond to salivary gland product, but to other thoracic factor(s). Intact thorax that was connected to 1-2 abdominal segments attracted considerably less mf than excised thorax. However, intact thorax attracted more mf than excised abdomen. Abdominal tissue size did not affect thorax attractiveness. Involvement of a large molecular weight protein(s) was suggested by the high attractiveness of the 100 kDa Centricon concentrator retentate and complete precipitation of attractant(s) by ethanol.

Animals↗
PubMed2001

A stochastic model for the aggregation of Onchocerca volvulus in nodules.

A model is presented which describes the aggregation of female Onchocerca volvulus in nodules and their distribution in the human population. The basic model is based on a single parameter, the formation probability q, which represents the probability with which incoming larvae form a new nodule. This parameter describes parasite behaviour which cannot easily be recognized in available data without modelling. The estimate for the average formation probability of muq = 0.39 suggests an attraction of the invading infective larvae to already existing nodules or resident worms with probability 0.61. No significant difference in muq was found between the forest and savanna parasite strains. The model can be used inversely to estimate the worm burden of persons from palpation data. The observed variance in the number of nodules per person requires the assumption of a variance-increasing mechanism which was implemented by heterogeneity within the host population (extended model with 2 parameters). Possible reasons for this heterogeneity are presented and its implications concerning the reproductive biology of the parasite are discussed.

Animals↗
PubMed2002

Neoplastic change in Onchocerca volvulus and its relation to ivermectin treatment.

A pleomorphic neoplasm (PN) is described from sections of Onchocerca volvulus worms in nodules excised from Cameroonian patients. PN is confined to older, non-fecund, female worms, and those classed as moribund/dead. It is mainly composed of small, roundish, basophilic cells of diverse sizes, often forming a 'rosette' pattern around amorphous eosinophilic centres. The cells have a high nuclear/cytoplasmic ratio and up to 2-3 mitoses/high-power field; some become grossly enlarged, highly polymorphic and contain large, irregular blocks of chromatin. The eukaryotic PN cells first appear posteriorly in the pseudocoelom, probably from ovarian cells; they spread anteriorly, invading or compressing the uteri. Ivermectin treatment increased the prevalence PN from 3.7% of 1422 female worms in 637 patients before treatment to 17.5% of 1134 worms in 511 patients after 3 years treatment. Ivermectin at 400-800 microg/kg annually, or at 150 microg/kg or 400-800 microg/kg 3-monthly, over 3 years, did not increase the PN prevalence significantly, as compared with standard doses of 150 microg/kg annually. In other small series of African patients, PN prevalence increased in worms 2, 4, 6 and 10 months after ivermectin treatment; but there was no increase after treatment with amocarzine, albendazole or diethylcarbamazine and suramin. PN may partly account for the increased macrofilaricidal action of ivermectin on female O. volvulus in patients treated for 3 years at 3-monthly intervals.

Adult↗
PubMed2003

The morphological discrimination of microfilariae of Onchocerca volvulus from Mansonella ozzardi.

There is no published account which allows the morphological discrimination of microfilariae of Onchocerca volvulus and M. ozzardi from each other. However, they occur together in parts of Brazil and Venezuela, and presumably there is always the possibility that migration could establish new sympatric populations in the future. The objective of this study was to evaluate simple morphological characters that might be used for species-diagnosis of microfilariae. The conclusions were that the location of microfilariae in the blood or skin, the body size and the nucleation of the nerve ring are expected to be useful first indications of species identity, but cannot be used for confident diagnosis. The structure of the cephalic armature (stained with alcian blue) seems to be species specific, but is of limited application because it is often difficult to see. However, the pattern of nucleation of the tail (as expressed by the ratio of the length of the terminal nucleus compared with the length of the tail space) is distinctive and is expected to be diagnostic.

Animals↗
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↗