Onchocerca lienalis microfilariae (mf) were cryopreserved in liquid nitrogen within skin-snips using methanol as a cryoprotectant and their viability evaluated and compared to mf cryopreserved free of host tissues using ethanediol as a cryoprotectant. Despite an initial delay in emergence, the methanol technique did not significantly affect the total numbers of mf emerging from skin-snips of various sizes (3·3–59·81 mg) compared to untreated controls over a 6 h period. Following thawing, the initial motility index (MI) scores of mf cryopreserved by either method were not significantly different from untreated controls; however, over a period of 15 days in culture the MI scores of both cryopreserved groups showed a small but significant overall decline, with the methanol technique producing the lowest scores. These changes in motility levels correlated with the numbers of mf which developed to the infective stage following intrathoracic injection into Simulium ornatum, although this ability to develop was a much more sensitive measure of parasite viability; compared to untreated control recoveries of 3rd-stage larvae, 63·9–71·7% (ethanediol technique) and 34·2–36·9% (methanol technique) of this number were recovered from cyropreserved groups. There were no significant differences in the lengths of infective larvae recovered from the insect heads from each treatment group, nevertheless there were higher numbers of 2nd-stage larvae recovered from the cryopreserved groups compared to the untreated controls. The methanol technique has the advantage of being easier to carry Out under field conditions, while parasite viability is significantly better using the ethanediol technique.

Use was made of seven FITC labelled lectins as tools to investigate the surface of Onchocerca lienalis larvae as they develop through to the infective third-stage in a natural vector, Simulium ornatum. The lectins were derived from Canavalia ensiformis (Con A), Lens culinaris (lentil), Triticum vulgaris (wheat germ), Arachis hypogaea (peanut), Helix pomatia, Phaseolus vulgaris (kidney bean) and Tetragonolobus purpureus (asparagus pea). Between 70 and 100 living parasites were examined for each developmental stage; i.e. skin microfilariae, late first-stages, second-stages, preinfective third-stages and infective third-stages isolated from the mouth parts of the flies. None of the lectins used bound to the surface of the microfilariae. However, progressive binding to the cuticle of the first- and second-stages was observed using Con. A, lentil lectin and wheat germ agglutinin (WGA). Following moulting to the third-stage, binding of these three lectins declined. Furthermore, as these lectins decreased, peanut and Helix pomatia lectins progressively increased in their binding, despite the fact that they showed little or no binding to the first- and second-stages; stages at which Con A, lentil and WGA were at their maximum. Asparagus pea and kidney bean lectins failed completely to bind to any of the larvae examined. Carbohydrate inhibition tests showed that the lectin was indeed binding specifically to glycoconjugates on the parasite surface. WGA binding was not inhibited by prior incubation with N-acetyl-D-glucosamine, even at high concentrations, but neuraminic acid did completely inhibit its binding. Judging from the patterns of binding on the nematodes themselves, the carbohydrates may not be vector in origin, but derive from the worms. The lectin specificities indicate that initially mannose/glucose type derivatives are present on the surface. Following moulting to the third-stage these are progressively replaced, or overlaid with galactosamine type derivatives, also present on the infective third-stage as it enters the bovine host. The availability of these surface glycoconjugates to attack mediated by natural insect lectins may be of importance in the parasite regulatory mechanisms of the blackfly. Variability in these surface carbohydrates, and in the response to them could well be a contributing factor in the cytospecific variation in S. damnosum susceptibility to geographical variants of O. volvulus.

Experiments were carried out to obtain additional data concerning the role of IgM antibodies, specific for the cuticular surface of the microfilariae (mf) of A. viteae, in clearing microfilaraemia from high-and low-responder mice infected by transplanted adult worms. Although BALB/c mice, which sustain a chronic microfilaraemia, produced IgM mf surface-specific antibodies, the binding to target mf was weak when compared to that of antibodies from the serum of the resistant C57BL/10 mice. Furthermore, antibodies from BALB/c mice were not as efficient as those from C57BL/10 mice in promoting the adherence of immune or control leukocytes to mf in vitro. Evidence is provided to show that mf shed surface bound antibody. Although the results do not establish conclusively the mechanism underlying the contrasting response phenotypes of C57BL/10 and BALB/c mice, they provide support for the involvement of antibody in controlling microfilaraemia and suggest that quantitative and qualitative differences in the amount and affinity of IgM antibody specific for the mf surface, together with the natural tendency of the mf to shed surface bound antibody at 37°C. may combine to allow the former strain to clear microfilaraemia efficiently whilst the latter sustains a chronic infection.

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.

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.

This paper uses simple mathematical models and statistical estimation techniques to analyse the frequency distribution of microfilariae (mf) in blood samples from human populations which are endemic for lymphatic filariasis. The theoretical analysis examines the relationship between microfilarial burdens and the prevalence of adult (macrofilarial) worms in the human host population. The main finding is that a large proportion of observed mf-negatives may be ‘true’ zeros, arising from the absence of macrofilarial infections or unmated adult worms, rather than being attributable to the blood sampling process. The corresponding mf distribution should then follow a Poisson mixture, arising from the sampling of mf positives, with an additional proportion of ‘true’ mf-zeros. This hypothesis is supported by analysis of observed Wuchereria bancrofti mf distributions from Southern India, Japan and Fiji, in which zero-truncated Poisson mixtures fit mf-positive counts more effectively than distributions including the observed zeros. The fits of two Poisson mixtures, the negative binomial and the Sichel distribution, are compared. The Sichel provides a slightly better empirical description of the mf density distribution; reasons for this improvement, and a discussion of the relative merits of the two distributions, are presented. The impact on observed mf distributions of increasing blood sampling volume and extraction efficiency are illustrated via a simple model, and directions for future work are identified.

Cats were repeatedly inoculated with infective larvae of Brugia pahangi. On parasitological grounds they could be divided into 5 groups. Group I – most cats (some 70%) became microfilaraemic (mf+) and retained high levels of microfilariae (mf) in their blood for over 2 years. In some Group I cats mf counts stabilized at high levels whilst in others mf counts continued to increase. Large numbers of fecund adult worms were recovered from their lymphatics. Adult counts were not made on the cats in the current experiments but over 100 adults have been recovered from ‘super-susceptible’ cats. Large amounts of B. pahangi adult antigen were consistently present in the serum of all Group I cats. About 30% of cats became amicrofilaraemic (mf–). In these cats the peak mf levels were seldom above 10000 mf/ml. Group II – these cats had less than 10 000 mf/ml and low antigen levels. After more than 1 year of being repeatedly infected B. pahangi adult antigen slowly declined and eventually could no longer be detected in their serum and the number of mf declined very slowly after the fall in antigen levels. This shows that in Group II cats the adult worms die and as the cats are resistant to the development of the continuing weekly inoculation of L3 no new adults can develop. Group III – these cats became mf – during the first year of infection but remained B. pahangi antigen-positive for many weeks after this and, at autopsy, had living adults in their lymphatics. Group IV – in these cats there was a sudden decline in the number of mf, usually in the first year and after they have received a relatively small number of re-infections. Circulating adult B. pahangi antigen disappeared from the serum within a short time of the loss of mf and at autopsy no adult female worms were found. Group V – these cats do not become mf+ when infected. These groups are compared to the parasitological groups seen in areas endemic for infection with Wuchereria bancrofti.

The susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to H2O2 and NO either in cell-free culture or from IFNγ-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 μM 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 IFNγ-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-TNFα 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 IFNγ-activated macrophages and in the case of B. malayi, killing is dependent on the generation of NO via TNFα.

After i.v. injection of 305 × 103 microfilariae (mf) per animal (50 g) into naive jirds, 50·8% of them could be recovered at autopsy 15 min later. Of these, 65·8% were calculated to be in the peripheral circulating blood (PCB) and were completely intact; 18·6% were recovered by perfusion of the lungs and 13·6% from the liver. In both organs about half the mf were associated with adherent lymphocytes and neutrophils but a few were partly disintegrated. Only 2·6% were recovered from the kidneys and the spleen. In long-term injection experiments using the same inoculum size the autopsy was done 15 min and 1, 3 and 6 weeks post-injection (p.i.) of mf into naive jirds. Throughout the experimental period the density of mf remained more or less constant in the PCB, but 3 weeks p.i. the density in the lungs increased up to 14 times to that in the PCB, whereas in the liver it decreased at the same time to a density similar to that in the PCB. In patent animals with adult worms delivering mf these were distributed as follows: 34·7% were calculated to be in the PCB; 24·4% were obtained by perfusion from the lungs and 22·0% from the liver; the rest were found in the kidneys (16·6%) and spleen (2·3%). In the lungs and the liver about 5/6 were associated with adherent cells, partly disintegrated or as fragments. In view of the fact that very few mf become disintegrated immediately after i.v. injection and also from their extremely long sojourn in the PCB, a low turnover rate of mf is presumed. The limited fecundity of the female adults combined with the 20 weeks median circulation time of injected mf in the PCB supports this view.

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.

Regular distribution of ivermectin reduces onchocerciasis transmission and morbidity by killing, within humans, the microfilarial stage of the parasite (microfilaricidal effect). In addition, ivermectin exerts a so-called embryostatic effect by which microfilarial production by the adult female worm becomes suppressed during a number of weeks after treatment. To assess the overall effect of ivermectin on onchocerciasis transmission and evaluate the likelihood of local elimination of the infection it is important to estimate the magnitude of the anti-fertility effect over the course of a treatment programme. Estimates of the effect of repeated drug treatments on the production of microfilariae by adult Onchocerca volvulus were obtained by developing a model that was fitted to data collected from three hyperendemic communities in Guatemala, where eligible residents received ivermectin twice per year for two and a half years. The data consist of microfilarial load measurements in the skin, collected just before each six-monthly treatment during the programme. The model that is developed describes the dynamics of an individual host's expected microfilarial load over the 30-month study period. We adopt a Bayesian approach and use Markov chain Monte Carlo (McMC) techniques to fit the model to the data. Combining estimates from the three villages, average microfilarial production in the first six months post-treatment was reduced by ~64% of its pre-treatment level, regardless of values chosen for the pre-ivermectin fertility rate within plausible ranges. Increased adult worm death rate after treatment (to mimic removal of macrofilariae via nodulectomy during the programme) resulted in a smaller estimated magnitude of the embryostatic effect (rate of microfilarial production was reduced by ~58% of pre-ivermectin value). After subsequent treatments, the rate of microfilarial production appeared to be similarly decreased. The data and analyses therefore do not support the hypothesis of a cumulative effect of multiple ivermectin treatments on microfilarial production by female worms.

Ivermectin treatment may induce severe adverse reactions in some individuals heavily infected with Loa loa. This hampers the implementation of mass ivermectin treatment against onchocerciasis in areas where Onchocerca volvulus and L. loa are co-endemic. In order to identify factors, including co-infections, which may explain the presence of high L. loa microfilaraemia in some individuals, we analysed data collected in 19 villages of central Cameroon. Two standardized skin snips and 30 μl of blood were obtained from each of 3190 participants and the microfilarial (mf) loads of both O. volvulus and L. loa were quantified. The data were analysed using multivariate hierarchical models. Individual-level variables were: age, sex, mf presence, and mf load; village-related variables included the endemicity levels for each infection. The two species show a certain degree of ecological separation in the study area. However, for a given individual host, the presence of microfilariae of one species was positively associated with the presence of microfilariae of the other (OR=1·79, 95% CI [1·43–2·24]). Among individuals harbouring Loa microfilariae, there was a slight positive relationship between the L. loa and O. volvulus mf loads which corresponded to an 11% increase in L. loa mf load per 100 O. volvulus microfilariae. Co-infection with O. volvulus is not sufficient to explain the very high L. loa mf loads harboured by some individuals.

An ultrastructural study of microfilariae of Wuchereria bancrofti was performed after in vitro treatment with diethylcarbamazine. One of the first morphological alterations produced by treatment with 5, 10 or 50 μg/ml of DEC was the loss of microfilarial sheaths. Drastic effects of DEC were only detected when microfilariae were exsheathed. Microfilariae treated with 5, 10 or 50 μg/ml DEC showed severely affected organelles, formation of several vacuoles mainly in the hypodermis, and cytolysis. Some microfilariae showed extreme cellular disorganization with abundance of electron-dense degenerating organelles, numerous large vacuoles, and nuclear condensation. Lamellar bodies probably related to an assembly of endoplasmic reticulum membranes were observed in some damaged microfilariae. Thus, in vitro treatment with concentrations of DEC similar to therapeutic conditions had direct effect by causing a loss of the microfilarial sheath of W. bancrofti with subsequent damage of organelles and apoptosis.

Lymphatic filariasis caused by the parasitic nematode, Brugia malayi, is a chronic human disease immunologically characterized by stimulation of Th2 cells and reduced antigen-specific T cell responses. Single stage intra-peritoneal infections with infective larvae (L3) or adult nematodes induce Th2 cells, while the microfilarial stage (Mf) stimulates IFN-γ and Mf-specific IgG1, IgG2a, IgG2b, IgG3 and IgM, but not IgE. To investigate whether IFN-γ is elicited by live Mf in their natural site of infection, mice were infected intravenously. Intravenous infection had a striking effect on the response to Mf and high levels of IgE were induced even in the presence of IFN-γ. Indeed IgE levels to Mf increased markedly with the number of immunizations, higher doses of Mf and prolonged exposure to Mf suggesting that under conditions of chronic antigen exposure, typical of human disease, Mf will stimulate high levels of IgE. The ability of Mf-induced IFN-γ to modulate or regulate a pre-existing Th2 response, was investigated by infecting mice initially with adult male worms to induce a Th2 response, followed 14 days later by infection with Mf. Although Mf stimulated IFN-γ in the presence of male adults, the antibody isotypes elicited did not reflect IFN-γ induction and IgG1and IgE dominated the response. Although it cannot be discounted that IFN-γ induction by Mf may act locally as an inflammatory mediator or modulator of Th2 cells, these data suggest that Mf-stimulated IFN-γ does not have a profound effect overall on progression of the Th2-dominated immune response to filarial infection.

The present study was undertaken in village Karondang in South-Sulawesi, Indonesia, to investigate the influences of genetic, household and environmental factors on Brugia malayi infection. Infection status was determined by measuring both microfilariae in night blood and anti-filarial IgG4, as a marker for detection of active filarial infection. A total of 171 residents participated in the study; familial relationships between subjects were registered to construct pedigrees and distances between households were measured. The data were analysed using a test statistic for familial aggregation. For distribution of microfilariae over the study population a genetic influence on infection susceptibility was favoured over the household and environmental effects. For anti-filarial IgG4, all 3 clustering models gave significant results, suggesting that genetic, household and/or environmental factors influence specific IgG4 antibodies.

Onchocercomata with a defined worm population were analysed to elucidate the distribution of mast cells. Nodules with live females were classified according to the presence or absence of microfilariae. Immunohistochemical staining was performed using antibodies specific for mast cells or IgE. Mast cells appeared singly or in diffuse accumulations perivascularly and in inflammatory infiltrates between adult Onchocerca volvulus and in the capsular area. No mast cells were detected in cystic parts. Only few, scattered mast cells were found in the fibrous zone around the adult worm. They were increased with stronger infiltration and hence, related to the inflammatory cells. Mast cells were never localized directly at adult worms or microfilariae. A correlation of the mast cell distribution to the occurrence of eosinophils was observed regarding higher numbers of mast cells and eosinophils in nodules with microfilariae-producing females. Nodules with single males revealed higher numbers of mast cells than nodules with non-producing females, although both contained very few eosinophils. Onchocercomata with dead worms contained significantly more mast cells than those with live filariae. In conclusion, the localization and frequency of mast cells is contingent on the vitality and productivity of the worms and therefore, indirectly and directly on the release of O. volvulus antigens.

Culex quinquefasciatus (Wiedemann) mosquitoes, which are refractory to Brugia pahangi (Buckley and Edeson) microfilariae, were fed simultaneously on Wuchereria bancrofti (Cobbold) and B. pahangi microfilariae. A selected stock of Aedes aegypti (L) which is susceptible to both of these filariae was used as a control. The exsheathment and migration rates of B. pahangi in C. quinquefasciatus increased significantly as compared to those of B. pahangi when given as a feed by itself. These two processes did not show any trend with time. On dissection, 12 and 14 days postinfection, 27% of C. quinquefasciatus were found to be infected with an average of two larvae per mosquito. Of the total number of larvae found 78% were W. bancrofti while 22% were B. pahangi.

Feeding C. quinquefasciatus on in vitro exsheathed B. pahangi microfilariae suspended in serum did not increase the rate of migration, whereas 71–100% of Ae. aegypti became infected with a mean of 3.5 larvae per mosquito.

Inoculation of C. quinquefasciatus mosquitoes into the thorax and abdomen with exsheathed B. pahangi did not increase their infection rate, whereas in Ae. aegypti 67–82% became infected.