Th2-biased inflammation with eosinophilia and IgE production is a hallmark of helminth infections. It is pronounced in hyperreactive onchocerciasis patients (‘sowda’ or ‘local form’), who efficiently kill microfilariae resulting in severe dermatitis and lymphadenitis. In contrast, hyporeactive patients (‘generalised form’) tolerate high microfilarial loads. This is thought to be mediated by regulatory CD4+ T cells and macrophages producing suppressive cytokines such as IL-10 and transforming growth factor-beta (TGF-β). We investigated whether hyperreactivity was reflected by lower local TGF-β production, analysing stable latent TGF-β1 expression in onchocercomas, lymph nodes and skin from hyperreactive and hyporeactive patients by immunohistochemistry. TGF-β expression was compared with that of IgE, IgG1, IgG4, and the antigen-presenting, CD4+ T cell-inducing MHC class II molecule HLA-DR. TGF-β was weakly and less frequently expressed by various cell types in onchocercomas, skin and lymph nodes from hyperreactive compared to hyporeactive patients. This applied to reactions around living and dead adult worms as well as dead microfilariae. Antigen-presenting cells strongly expressed HLA-DR in both forms, but their numbers were reduced in hyperreactive nodules. Plasma cells produced more IgE and IgG1, but less of the anti-inflammatory antibody IgG4 in hyperreactive onchocercomas. In conclusion, hyperreactivity is linked with reduced local expression of TGF-β, HLA-DR and IgG4, which might contribute to the insufficient down-regulation of inflammation via TGF-β- and HLA-DR-induced regulatory lymphocytes.

Introduction. Several reports indicate high prevalences of both onchocerciasis and epilepsy in some regions of Africa. This raises the question of whether these diseases are associated. We therefore investigated people with epilepsy and/or onchocerciasis living in an area in Tanzania endemic for Onchocerca volvulus (O. volvulus). Methods. We collected clinical information, skin snips, and blood from 300 individuals, and cerebrospinal fluid (CSF) from 197. Participants were allocated to 4 groups consisting of people with epilepsy and onchocerciasis (n=135), those with either epilepsy (n=61) or onchocerciasis only (n=35), and healthy individuals (n=69). Samples were evaluated for microfilaria, IgG4 antibodies against O. volvulus, O. volvulus antibody index (CSF/serum), and CSF routine parameters. Polymerase chain reaction (PCR) was performed on skin snips and CSF. Results. No difference was found in microfilarial density between participants with and without epilepsy (P=0·498). The antibody index was raised in 2 participants. CSF PCR was negative in all samples tested. Discussion. Our results do not give evidence of a relationship between O. volvulus and epilepsy. Despite the fact that 2 participants had raised antibody index, the existence of cerebral onchocerciasis caused by migration of microfilariae into the CSF appears unlikely. However, to date unexplored reactions to the infestation with O. volvulus causing epilepsy cannot be excluded.

Up to 5% of untreated female Onchocerca volvulus filariae develop potentially fatal pleomorphic neoplasms, whose incidence is increased following ivermectin treatment. We studied the occurrence of 8 filarial proteins and of Wolbachia endobacteria in the tumor cells. Onchocercomas from patients, untreated and treated with antibiotics and anthelminthics, were examined by immunohistology. Neoplasms were diagnosed in 112 of 3587 female and in 2 of 1570 male O. volvulus. The following proteins and other compounds of O. volvulus were expressed in the cells of the neoplasms: glutathione S-transferase 1, lysosomal aspartic protease, cAMP-dependent protein kinase, alpha-enolase, aspartate aminotransferase, ankyrin E1, tropomyosin, heat shock protein 60, transforming growth factor-beta, and prostaglandin E2. These findings prove the filarial origin of the neoplasms and confirm the pleomorphism of the tumor cells. Signs indicating malignancy of the neoplasms are described. Wolbachia were observed in the hypodermis, oocytes, and embryos of tumor-harbouring filariae using antibodies against Wolbachia surface protein, Wolbachia HtrA-type serine protease, and Wolbachia aspartate aminotransferase. In contrast, Wolbachia were not found in the cells of the neoplasms. Further, neoplasm-containing worms were not observed after more than 10 months after the start of sufficient treatment with doxycycline or doxycycline plus ivermectin.

The antibody responses of 8 cattle experimentally infected with Onchocerca ochengi to 18 recombinant O. volvulus antigens were measured by ELISA. In addition to establishing antigenic cross-reactivity between the species, the dynamics of antigen-specific responses were examined to assess how the recognition of the antigens compared to the known stage-specificity of expression. Six cattle responded to all of the antigens and 2 animals responded to all but 1. The dynamics of the recognition of 4 antigens (B20, MOv-2, MOv-14 and OvNHR2 02E1) were characterized by rapid seroconversion following infection. Antibody levels to 2 antigens (Ov7 and OvALT-1) increased gradually over the course of infection. Antibody levels to 4 antigens (OvTPX-2, OvL3Chitinase, Ov103 and Ov9m) reached maximum levels coincident with the onset of patency. The levels to 3 antigens (OvProalf C50, OvAldolase, Ov39) varied little over the course of infection. Responses to antigens with functional similarities (OvSOD1, OvSOD2 and OvSOD3 or OvGST1 and OvGST2) showed comparable temporal profiles. This study demonstrates the high degree of immunological cross-reactivity between the antigens of O. volvulus and O. ochengi. The immunogenicity of antigens varied over the course of infection in an antigen-specific manner, which not always reflected developmentally regulated expression of the corresponding gene, possibly owing to cross-reactive epitopes on distinct parasite products.

Chromosomes of Nigerian Onchocerca volvulus were compared with those of Guatemalan O. volvulus. Both parasites had basically the same chromosomal construct (2n=8, XY type). Autosomes consisted of a pair of large and two smaller pairs. Sex chromosomes were made up of medium sized X chromosome and very small Y chromosome. It was not possible to infer the position of the centromeres.

Comparative morphological and biometric characteristics of microfilariae of Onchocerca gutturosa and O. volvulus from different geographical areas (Upper Orinoco, Venezuela; Togo; Liberia) were assessed. “Stepwise” discriminant analysis and Mahalanobis estimators were applied to measure distance between populations. The results indicate a strong similarity between the two strains from the Upper Orinoco (Venezuela) and the Togo strain, as well as a clear separation between these strains and that of O. gutturosa. The Liberian strain was easily distinguishable from microfilariae from Togo and Venezuela. Discriminant analysis showed the Liberian deme to be as different from the Venezuelan and Togo demes as these demes were from microfilariae of the reference species, O. gutturosa. Although it is necessary to confirm these data using formalin-fixed specimens obtained from the skin, the present findings suggest the existence of geographically-different strains of O. volvulus in America and Africa.

Infective larvae of Onchocerca lienalis and O.volvulus implanted subcutaneously within micropore chambers into laboratory hosts moulted to the fourth stage (L4) and underwent limited development and growth. Similar recoveries of O. lienalis L4 larvae in the range of 33–66% were obtained from chambers implanted into CBA and BALB/c strains of mice, jirds, and the natural bovine host. A relatively constant proportion of larvae survived up to 24 days post implantation and thereafter recoveries declined, although some worms were still alive after 96 days. Recoveries of O. volvulus L4 larvae from chambers given to normal or T-cell deprived mice were equivalent to one another and to those obtained with O. lienalis. Moulting of O. lienalis in chambers was observed on days 3 and 5, in close accordance with the timing of the third moult in cattle following systemic infection. Moulting of O. volvulus occurred between days 3–6. Morphological changes in developing larvae included a small but significant increase in length, a transient increase in width, and early development of the spicular primordia and genital tube. L4 larvae of O. lienalis, but not those of O. volvulus, exhibited 3 distinct caudal papillae not present on infective larvae.

A probable case of vertical transmission of Onchocerca volvulus microfilariae in Dass Bauchi State, Nigeria is presented. A 28-week-old male child delivered by a 39-year-old Fulani woman suffering from onchocerciasis was positive for O. volvulus microfilariae based on skin snip examinations. The child had general pruritic rash and intermittent fever. Although the child's skin snip on re-examination five weeks later was positive, the fever had gradually subsided.

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.

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 µm 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 cuticle were identified.

Comparison of glycolysis in Brugia pahangi and Onchocerca volvulus by C 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 δ-aminobutyrate shunt to produce succinate which is then further metabolized to acetate and carbon dioxide as end-products.

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 Mr 12–42 kDa. In the immunized and non-patent chimpanzee, by contrast, serological recognition of uterus-derived OvAg with Mr 14 kDa and 105 kDa disappeared by 19 months post-challenge (p.c.). During immunization, Acanthocheilonema viteae L3 antigens of Mr 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-γ alone, and IFN-γ 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-γ, 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.

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 weasel-transmitted O. volvulus from Uganda.

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. Amicrofilerdermic (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.

In order to construct an analytical model of onchocerciasis transmission, it is necessary to elucidate the functional relationships of the various population rate processes taking place within the human and vector hosts. Two previous papers have explored the evidence for density-dependent regulation in relation to microfilarial intake by, and larval development within, the Simulium host. This paper investigates the survivorship of wild-caught blackfly samples fed on subjects with different intensities of Onchocerca volvulus microfilarial infection. Analyses were based on data for Guatemalan S. ochraceum s.l. (possessing a well-developed cibarial armature), West African S. damnosum s.l. (forest species), and South Venezuelan S. guianense (the latter two lacking a toothed cibarium). The mean survival times of samples of the 3 species, kept under laboratory conditions, decreased as parasite intake increased, the rate of mortality being dependent on the fly’s age (measured as time post-feeding) and on the worm load acquired. An empirical, timedependent hazard function was fitted to observed death rates/fly/day which rose very shortly after engorgement, declined subsequently, and rose again throughout the extrinsic incubation period of the parasite. The parameters of this hazard model were all positively correlated with the density of microfilariae in the bloodmeal. Expressions of survivorship and life-expectancy as explicit functions of time post-feeding and mean parasite intake were derived. The average expectation of life at engorgement for uninfected flies in the laboratory was estimated to be around 1 week for both, armed and unarmed blackflies. Residual life-expectancy decreased with time post-feeding and microfilarial load in both categories of vectors. This decline (resulting from age- and parasite-dependent mortality rates) was much more pronounced in those species lacking a toothed fore-gut. Whilst a fraction of heavily infected S. ochraceum was able to survive the latent period of the parasite, being therefore potentially capable of transmitting the infection, equivalent worm loads in S. guianense resulted in a drastic reduction of the expectation of infective life. These results provide additional evidence to support the hypothesis that, in the case of intrinsically susceptible vectors, unarmed simuliids are more efficient at low microfilarial loads, when the transmission rate from human to vector host is higher, and parasite-induced fly mortality is negligible. The opposite takes place in armed flies, which perform poorly at low parasite burdens and better at heavier loads, with little parasite-induced vector death.

The transmission success of Onchocerca volvulus is thought to be influenced by a variety of regulatory or density-dependent processes that act at various points in the two-host life-cycle. This paper examines one component of the life-cycle, namely, the ingestion of microfilariae by the simuliid vector, to assess the relationship between intake of larvae and the density of parasites in the skin of the human host. Analysis is based on data from three areas in which onchocerciasis is endemic and includes published information as well as new data collected in field studies. The three areas are: Guatemala (Simulium ochraceum sl.), West and Central Africa (savanna members of the S. damnosum complex), and South Venezuela (S. guianense). The data record experimental studies of parasite uptake by flies captured in the field and fed to repletion on locally infected subjects who harboured varying intensities of dermal microfilarial infection. Regression analyses of log transformed counts of parasite burdens ingested by the flies plotted against log transformed counts of microfilariae per mg of skin revealed little evidence for saturation in parasite uptake by the flies as the intensity in the human host increased. There was a positive and highly significant rank correlation between both variables for the three blackfly species. In an alternative analysis a model was fitted to data on prevalence of flies with ingested microfilariae (mff) versus dermal mean intensities. The model assumed an overdispersed distribution of the number of mff/fly and a given functional relationship between intake and skin load. The results of both approaches were consistent. It is concluded that parasite ingestiossssn by the vector host is not strongly density dependent in the three geographical areas and ranges of dermal loads examined. It therefore appears that this transmission process is of reduced importance as a regulatory mechanism in the dynamics of parasite population growth.

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. Treatment of these animals with CGP 20376, with autopsy 2 weeks later, resulted in a small macrofilaricidal effect (28% worm death). Some reduction in the motility of living worms recovered from CGP 20376-treated gerbils was also evident. Good survival of control worms recovered 2 weeks post-implantation suggests that male O. volvulus might survive longer subcutaneously. The prospects for undertaking meaningful drug evaluations against O. volvulus in vitro (and in vivo in rodents) is therefore promising, but because of parasite availability will probably be limited to those compounds which have shown potential in other filarial test systems.

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, α1-antitrypsin and α1-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.

Antigens were extracted from the epicuticle/cuticle of intact female Onchocerca volvulus using 2% 2-β-mercaptoethanol and 1% SDS. In Western blot analysis a human infection serum selected for its high antibody titre against whole worm homogenates did not recognize any component solubilized by 1% SDS. However, the same serum did bind at least 7 antigens among the material extracted with 2-β-mercaptoethanol. These antigens have apparent molecular weights (Mr) of: 15 000, 18 000, 28 000, 78 000, 98 000, 120 000 and 200 000. In ELISA using this preparation as target antigen, 151 out of 153 human infection sera gave positive results. An Onchocerca-specific IgG1 monoclonal antibody, designated Cam1, recognized the 28 000 Mr antigen, which is the most prominent antigen detected by Western blot analysis using human infection sera. In ELISA, using material affinity-purified with Cam1 as target antigen, 149 out of 153 human infection sera gave a positive IgG response. From a cDNA library three expressing clones were isolated with a rabbit serum raised against 2-β-mercaptoethanol solubilized material. One of these clones was recognized by the monoclonal antibody Cam1.

A previous paper reported that the intake of Onchocerca volvulus microfilariae (mff) by different species of Simulium is essentially proportional to the parasite load in the skin of infected carriers. This paper examines the fate of the ingested mff in susceptible vectors to assess the relationship between parasite intake and infective larval output in blackfly species with and without well-developed cibarial armatures. Analysis is based on data from 3 onchocerciasis endemic areas: Guatemala (S. ochraceum s.l.), West Africa (S. damnosum s.l./S. sirbanum) and the Amazonian focus between South Venezuela and Northern Brazil (S. guianense and S. oyapockense s.l.). The data, which include published and unedited information collected in the field, record experimental studies of parasite uptake by wild flies maintained in captivity until the completion of the extrinsic incubation period. The relationship between L3 output (measured as the mean number of successful larvae/fly or, as the proportion of flies with infective larvae) and average microfilarial intake, was strongly non-linear. This non-linearity was best represented by a sigmoid function in case of armed simuliids (S. ochraceum s.l., S. oyapockense s.l.), or by a hyperbolic expression in that of unarmed flies (S. damnosum s.l., S. guianense). These results are compatible, respectively, with the patterns of ‘initial facilitation’ and ‘limitation’ described in culicid vectors of lymphatic filariases. A maximum mean number of 1–3 L3/fly was observed in all 4 vectors. It is concluded that O. volvulus larval development to the infective stage is regulated by density-dependent mechanisms acting at the early phase of microfilarial migration out of the blackfly's bloodmeal. Damage by the bucco-pharyngeal armature may also be density dependent. A hypothesis, based on this density dependence is forwarded to explain initial facilitation, so far only recorded in vectors with well-developed cibarial teeth. Our results provide quantitative support for the conjecture that chemotherapy alone is likely to have a greater impact on reducing onchocerciasis transmission in endemic areas where the main vector has a toothed fore-gut than in foci where the vectors have unarmed cibaria.