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River blindness is a seriously debilitating disease caused by the filarial parasite Onchocerca volvulus, which infects millions in Africa as well as in South and Central America. Research has been hampered by a lack of good animal models, as the parasite can only develop fully in humans and some primates. This review highlights the development of two animal model systems that have allowed significant advances in recent years and hold promise for the future. Experimental findings with Litomosoides sigmodontis in mice and Onchocerca ochengi in cattle are placed in the context of how these models can advance our ability to control the human disease.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.

Onchocerca microfilariae were isolated form the umbilicus and neck of 438 cow hides at the abattoir in Tübingen, F.R.G. The overall Onchocerca infection rate was 40.4%. The presence of Onchocerca lienalis and O. gutturosa microfilariae, which are difficult to distinguish by morphological criteria, was retrospectively demonstrated after artifically infesting Simulium ornatum and Culicoides nubeculosus and identifying the infective larvae recovered. Nine of 16 samples of umbilical microfilariae fed to C. nubeculosus through a latex membrane developed to O. gutturosa third stage larvae (L3). Six of seven umbilical samples injected into the thorax of S. ornatum yielded O. lienalis L3. In six infestation trials in which microfilariae were introduced both into S. ornatum and C. nubeculosus, O. lienalis L3 were recovered exclusively from simuliids, while O. gutturosa L3 developed only in midges. Of six umbilical skins tested by cross-infestation, one contained exclusively O. gutturosa microfilariae, four only O. lienalis microfilariae and one was infected with both species. Developmental success of O. lienalis microfilariae to L3 in S. ornatum following intrathoracic injection was 22% of the mean inoculum. O. gutturosa microfilariae, ingested by C. nubeculosus through a latex membrane, developed to L3 at a rate of 2.3% of the mean microfilarial uptake.

A new technique for assessing vital and degenerative microfilariae (mf) in different skin layers of onchocerciasis patients is described. Vertical sections at least 5 mm deep were collected from the surgical nodulectomy wounds before and 2, 4 and 28 days after treatment with ivermectin and fixed in ethanol. Skin biopsies were punched horizontally with trephines and digested enzymatically with collagenase. In untreated biopsies 80% of the mf were located in the uppermost layer of 1 mm and only 1.2% were degenerated. After treatment with ivermectin the total number of mf was significantly reduced, and the distribution of living and remnant mf in the different skin layers had changed. The proportion of degenerated mf had increased markedly, but did not exceed 6% of the total pre-treatment mf level, i.e., the vast majority of the mf had actually disappeared from the skin. It is suggested that ivermectin reduces microfilarial motility slightly, and that mf are then attacked by adhering immunocompetent cells of the host and cleared by the lymphatic drainage.

This study compiles observations on the reproductive capacity of O. volvulus. Adult parasites enzymatically isolated from excised onchocercomata of untreated and chemotherapeutically treated patients, and from inhabitants living in areas with long vector control, were investigated to assess their fecundity. Changes of microfilaria development in utero and microfilaria release were assessed or estimated after treatment of patients with micro-filaricidal drugs that interfered with the development of intra-uterine stages. Intra-uterine production of microfilariae: After treatment of patients with ivermectin a daily development of 2500 to 4000 uterine microfilariae per female worm was observed. Actual output of microfilariae: Microfilariae left actively the female worms. The daily microfilaria release in vivo was 700 to 900 microfilariae, assessed after treatment of patients with mebendazole. In vitro most worms isolated from untreated patients shed between 500 to 1500 microfilariae per day.-The microfilarial load of 56 adult patients calculated from microfilarial skin counts was 12 million on the average. Taken for granted a mean life span of a microfilaria of 1.0 to 1.5 years, 22 to 47 female worms per patient would suffice to maintain this microfilarial load on a constant level. Excision of all palpable nodules showed a geometric mean of 15.9 female worms in these patients. It is suggested that factors intrinsic in the host and the adult worms partially operate together to regulate and maintain a stable microfilarial density.

A technique for the isolation of adult Onchocerca volvulus from excised onchocercomata is described. The nodules are incubated in medium 199 containing 1-5 mg collagenase and 0.2 mg gentamicin per ml for 6-48 hours in a waterbath at 30-37 degrees C. A proportion of the worms can be isolated alive.

The staining patterns of acid phosphatase activity of the various larval stages of Onchocerca volvulus from experimentally infected Simulium damnosum s.l. have been described. The enzyme activity was localized in the anal vesicle/pore area of the first-stage larva, in the anal vesicle and intestine of the secong-stage larva and all over the body in the third-stage larva. Early third-stage larvae could be differentiated from late third-stage infective larvae by the presence of pronounced enzyme activity in the rectum area of the former. There were no detectable differences in the staining patterns between the forest and Sudan-savanna strains of O. volvulus developing in S. damnosum.

A technique for the assessment of the total number of microfilariae in skin snips from onchocerciasis patients is described. For the digestion of the skin tissue the biopsies are incubated at room temperature for 24 hours in medium 199, containing 3 mg/ml collagenase and antibiotics. This process leaves the microfilariae unharmed. A comparative study of the emergence of the microfilariae in different media showed that only 20% of the total number were released after 30 minutes in distilled water. After 24 hours incubation in isotonic saline 80% were found.

The collagenase technique developed more than twelve years ago has successfully been applied to isolate living and intact O. volvulus from excised onchocercomata for various biological, morphological, biochemical, immunological and in vitro studies. The experience gained with this technique and its modifications is critically reviewed giving practical advice for the selection, preservation and treatment of nodules for different purposes, especially for the evaluation of antifilarial drug effects. The composition of the worm load, the morphology of adult worms found in untreated patients, signs of degeneration and ageing of worms are described to show the broad morphological spectrum of the worm population in endemic areas to avoid false conclusions in drug trials. When these parameters are carefully considered, the collagenase technique provides a reliable method for examination of onchocercomata. An immediate answer can be obtained concerning the viability of the parasite. The qualitative and quantitative assessment of reproduction (embryogram) can give further valuable information on any changes in the reproductive dynamics and provide a sensitive indicator of any disturbances in the adult worms which may explain the early or late reinvasion of microfilariae into the skin after treatment with microfilaricidal drugs.

Onchocerca tarsicola is a subcutaneous filariid of the red deer living on the tendons of the radial-carpal and tibial-tarsal joints. Its microfilariae concentrate mainly in the external ears. The extrinsic cycle of this parasite was investigated. In a hyperendemic area wild-caught Odagmia ornata were dissected, as well as samples which had fed on a tame deer and been kept in the laboratory after the infecting blood-meal. About 3 to 5% of the flies were infected with larval stages of Onchocerca tarsicola. More than 40% of Odagmia ornata induced to feed on the ears of the deer became infected and about 30% of the flies which survived more than 18 days contained third-stage larvae. The larval development in the vector is described. Prosimulium nigripes appears to be a second natural vector of Onchocerca tarsicola. Observations concerning the potential vectors of O. flexuosa, O. tubingensis and Cutifilaria wenki living in red deer are discussed.

The midbody regions of female worms of six Onchocerca species (O. flexuosa, O. tarsicola, O. lienalis, O. gutturosa, O. armillata, O. gibsoni) were studied by transmission electron microscopy. The cuticular layering was rather similar in all species with the ridges built up by the cortical layers and the inner cuticular striations by the median or basal layers. Differences in the epicuticular morphology were considerable. O. flexuosa and O. lienalis had a thin epicuticle without protuberances, the epicuticle of O. armillata carried small knobs, and O. tarsicola, O. gutturosa, and O. gibsoni had a thick trilaminar epicuticle with long protuberances. Extreme hypertrophy of hypodermis and reductions of somatic musculature were observed in O. flexuosa and O. gibsoni. Less extended thickenings of the hypodermis were observed in the other species. No degenerative alterations were found in the muscle cells of O. gutturosa and O. lienalis. The intestinal lumen of most of the species was in a central position, but in O. tarsicola and O. gibsoni the lumen was reduced to small clefts between the intestinal cells. In these species, numerous electron-dense, concentric granules were observed in the cytoplasm of the intestinal cells. The proportions of the various organs differed considerably from species to species, e.g., the uteri contained the embryos filed one behind the other in O. tarsicola, whereas 50 or more embryos were found beside one another in cross-sections of the uterus of O. gibsoni. The comparative study showed that O. gibsoni and O. volvulus have many derived morphological characteristics in common and that in the other species more primitive stages of development of these morphological marks can be observed.

Description of O. garmsi n.sp. in the connective subcutaneous tissue of Cervus elaphus; furhter information on the description of O. tubingensis Bain and Schulz-Key, 1974, in subcutaneous nodules of the same host.

In the Liberian rain-forest and the savanna of Upper Volta 189 adult nodule carriers were operated on. From 2231 extirpated nodules 3327 male and 5713 female macrofilariae were isolated. About 98% of the male worms and 88% of the female worms were found alive. The sex ratio of the live male and female worms was 1:1.5 in Liberia and 1:1.6 in Upper Volta. Less than 1% of all live macrofilariae were immature in both countries. 22% of the live male worms in Liberia were regarded as old. The percentage of old male and female worms in Upper Volta and that of the old female worms in Liberia were similar (5-7%). The portion of old worms was independent of the age of the patients in Liberia. Dead worms were found in 66% of the Liberians and in 85% of the Voltaics. The percentage of patients with dead worms increased with the age. About 0.5% of all male and 8% of all female worms were calcified. In Liberia the percentage of calcified worms increased with the age of the patients. In both countries the highest number of live and dead worms were found on the pelvic girdle. On the thorax many more macrofilariae were found in Upper Volta than in Liberia. This may contribute to the severe eye lesions in this savanna area. The presented findings provide some basic information for the planning and performance of trials with macrofilaricidal drugs in hyperendemic areas of West Africa. However, the striking differences between individual worm burdens have to be considered.

Adult Onchocerca volvulus were enzymatically isolated with collagenase from excised nodules and kept in TC medium 199 with Hank's salts supplemented with various sera. Male and female worms survived in the culture medium and 10% human serum on average for about 11 days (maximum 28 days) and 14.5 days (maximum 42 days). Up to 4,000 microfilariae were expelled by each female per day, but the production of new oocytes or development of embryos could not be observed in vitro.

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.

Females of O. volvulus show several typical morphological alterations during their ontogenesis. In order not to attribute such natural findings to the efficacy of drugs in drug trials female worms isolated from untreated patients in Liberia and Togo were examined by light microscopy. Local incrustations could be found regularly on the surface of the female parasite. Different inclusions are described which could often be found. They partially lead to degeneration and calcification. For the evaluation of ovocidal or embryocidal efficacy of drugs the developmental stages of 290 untreated female worms were quantitatively assessed, normal and pathologically altered forms were distinguished. Embryos were obviously not produced continuously but in intervalls of unknown length and frequency. Two third of the females contained embryos and microfilariae, one third showed eggs only or had empty uteri. The number of the developmental stages per female varied considerably. On the average more than 200,000 stages per female were found.

Adult Onchocerca voluvlus and infective larvae, but not microfilariae contain an immunodominant antigen (33,000 and 21,000 Mr in females, 39,000, 33,000, and 21,000 Mr in males, 133,000 Mr in infective larvae) which is recognized by an Onchocerca-specific mAb. The component is part of the reproductive organs and muscles. 96.2% of onchocerciasis sera contained antibodies detectable by immunoblotting against it. Antigen purified by immunoaffinity chromatography was specifically recognized in immunoblots by onchocerciasis sera, but not by sera from other filarial infections. The high immunogenicity, the specificity, and the occurrence in infective larvae of this antigen indicate an immunodiagnostic potential and a possible role in the immunobiology of the parasite.

The biology of O. flexuosa was studied during the examination of numerous subcutaneous nodules, which were taken from the hides of red deer of all age-classes. In fawns the development of the nodule is induced by the immature stages, probably by the females. At the same time a hematome is formed. Males of different ages penetrate into the nodule where mature or immature females are present. Later they mate and remain within the nodule until the microfilariae in the uteri of the females develop. Usually 1 or 2, occasionally 4 females are found together. During the development of the embryos in the uteri the initially stretched female winds up. Only a short anterior part remains always straight. It is put out through the wall of the capsule in order to release the microfilariae. The males are often the majority reaching to 13 in one nodule. During the release of the microfilariae the males are found outside the nodule on the surface or near-by in the subcutaneous connective tissue. Both sexes are able to mate a second time. In older deer the males are also able to induce the development of nodules.

In a village hyperendemic for onchocerciasis in Burkina Faso, where the vector had been controlled by the Onchocerciasis Control Programme since 1976, 65 patients were treated with doses of 37 to 71 mg/kg suramin in 1979. The viability and fertility of the adult parasites were studied in 217 nodules excised from 42 patients one month to four years after treatment using the collagenase technique. Most of the worms had survived the treatment. Male worms were more susceptible to suramin than female worms. The development of intrauterine stages initially continued, but was completely suppressed after several months. The female worms remained sterile in those patients who had received more than 60 mg/kg suramin. In other ones 11 and 13% of the female worms showed new embryonic stages in the uteri during two follow-up examinations in the second year. In a final examination in 1983 the reproduction had declined again. However, the reproductivity of the superannuated worms had considerably decreased in the untreated patients as well. All patients had shown a very high microfilardermia in 1979. After treatment the microfilarial densities dropped to levels near zero depending on the dose of suramin administered, but in none of the patients was the reduction complete during the first year of the treatment. Microfilariae gradually reappeared in the second year in several patients who had received less than 60 mg/kg suramin. In the final follow-up in 1983 most of the treated patients had become negative in skin counts, although they still harboured viable worms.(ABSTRACT TRUNCATED AT 250 WORDS)

The application of a new technique is described permitting a better recognition of activities of drugs on macrofilariae of Onchocerca volvulus. In two villages of a hyperendemic focus in Liberia onchocercomata were excised from untreated persons and from patients treated with conventional doses of suramin or metrifonate. The adult worms were enzymatically isolated with collagenase and microscopically examined for degenerative alterations including calcifications and for the presence of eggs and embryos in the female parasites. Six weeks after the last dose of suramin the degenerative alterations could not be distinguished qualitatively from those in untreated worms, but a statistically significant macrofilaricidal efficacy could be shown by quantitative analyses, although more than half of the parasites were still alive at this early date. Metrifonate did not show any macrofilaricidal effect.

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.

During a study on the effects of nodulectomy on onchocerciasis in a village in the Liberian rain-forest 400 Onchocerca-nodules were excised from 34 inhabitants. The analysis of the nodules revealed a worm burden of 718 complete live male and 1062 complete live female worms as well as 9 male and 117 female worms which were mostly calcified and degenerating. 73% of the live females contained developing embryos or released microfilariae, the other females either contained eggs or showed empty uteri. Some observations were made on degenerating worms. The male:female sex ratio was 1:1.5 and possible reasons for this are discussed.

In an attmept to find an experimental vector for Onchocerca volvulus which can be easily be bred under laboratory conditions, the susceptibility of Anopheles gambiae and Aedes aegypti to infection with O. volvulus was studied. The mosquitoes were fed on a membrane feeding apparatus containing microfilariae of O. volvulus. Whereas A. gambiae was refractory to infection. 14.9% of A. aegypti females became infected with developing stages, 11,2% showed young and mature third-stage larvae as well as some which had developed abnormally. However, no infective larvae of O. volvulus were found in the abdomen or the head of the mosquitoes.

Onchocerciasis, or river blindness, is caused by infection with Onchocerca volvulus, a filarial parasite which infects about 40 million people in Africa and Latin America. Epidemiological, clinical, entomological and serological studies of African onchocerciasis led to the hypothesis that Onchocerca volvulus exists in different forms in the forest and savannah. It is uncertain if these differences are due to genetic differences within O. volvulus itself, or to epigenetic factors, such as differences in the host populations. To date no basic biochemical differences between the forest and savannah populations of O. volvulus has been found, although isoenzyme studies have shown that differences in allele frequency between forest and savannah populations exist. Here we describe the isolation of a DNA sequence that seems to be specific for the forest form of O. volvulus, the first indication of a basic genetic difference between the savannah and forest forms.

O. garmsi is a filaria living free in the subcutaneous tissues on the chest of red deer. The microfilarial distribution pattern in the skin of the host is studied. The convergent distribution of microfilariae of O. garmsi, O. tubingensis and O. cervicalis on the one hand and of O. tarsicola and W. rugosicauda on the other hand is discussed.

Lactate dehydrogenase and malate dehydrogenases were partially purified and characterized from adult O. volvulus. The molecular weight of lactate dehydrogenase was determined to be 130 000, those of malate dehydrogenase I and II to be 120 000 and 65 000, respectively. The activities of both malate dehydrogenases and of the lactate dehydrogenase were strongly inhibited by suramin. The inhibition constants were determined to be in the range of 2 microM to 5 microM. The type of inhibition was found to be competitive with respect to the coenzyme NADH and to be non-competitive to the substrates. It is suggested that the mode of action of suramin in the therapy of Onchocerciasis might depend on the blokkade of reoxidation of NADH produced within the glycolytic pathway.