Development of Onchocerca volvulus (Filarioidea: Onchocercidae) in the West African black fly Simulium yahense (Diptera: Simuliidae) in Liberia.

Simulium yahense black flies infected with microfilaria of Onchocerca volvulus were kept in a defined insectary environment in Liberia, West Africa. A daily sample of infected flies was dissected for larvae developing in the thoracic muscles and examined for growth in stadial development. Microfilariae ingested by black flies transformed to the L1 larval stage without molting. Successive larval development included molting to the L2 stage and, finally, to the L3 stage, which was infective in humans. The cephalic cap, consisting of a laterally located hook and central stoma, occurs in the first larval stage. The caudal appendix and the laterally located anal opening are apparent in the L1 larva. In the L2 stage, the cephalic cap is lost and the large circular stoma becomes surrounded with elevated flaps. The caudal appendix was lost after larvae molted to the L3 stage, and in its place, 3 terminal papillae developed. Sense organs, such as 2 opposing phasmids and 8 papillae that were arranged into 2 circles, developed in the cephalic region of the L3 larva. The evidence of pathological consequences due to the presence of the L3 larva in the fly host are illustrated and discussed.

Brugia malayi microfilariae (Nematoda: Filaridae) enhance the infectivity of Venezuelan equine encephalitis virus to Aedes mosquitoes (Diptera: Culicidae).

We examined the potentially conflicting effects that microfilarial (MF) enhancement of viral infectivity and MF-induced mortality in mosquitoes have on the vectorial capacity of Aedes aegypti (L.), Aedes triseriatus (Say), and Aedes taeniorhynchus (Wiedemann) for Venezuelan equine encephalitis virus (VEE) when mosquitoes feed on gerbils co-infected with Brugia malayi (Buckley). Groups of mosquitoes were fed on gerbils that were either dually infected (VEE plus B. malayi MF) or singly infected (VEE only). Mosquito mortality was recorded daily, and 5-8 d later, surviving mosquitoes were assayed for disseminated viral infection. The contrasting effects of MF enhancement and MF-induced mortality differed among mosquito species and were determined by the nature and consequences of MF penetration through the mosquito midgut, but not to differences in mosquito susceptibilities to parenterally introduced virus. In Ae. aegypti, MF-induced mortality was high and tended to eliminate any significant effect of MF enhancement. In Ae. triseriatus, MF-induced mortality was low, and feeding on dually infected hosts resulted in 9 times as many mosquitoes with disseminated viral infections as did feeding on singly-infected hosts. In Ae. taeniorhynchus, MF-induced mortality was extremely high, yet under our experimental conditions, feeding on a dually infected hosts resulted in nearly 30 times as many disseminated infections as did feeding on singly infected hosts. The final outcome on vectorial capacity depended on the specific combination of MF, virus, and mosquito species involved. Therefore, future efforts toward understanding MF enhancement should be directed toward mosquito-virus-parasite species combinations that occur together in nature.

The Johns Hopkins University School of Hygiene and Public Health, Lloyd E. Rozeboom mosquito collection.

The Lloyd E. Rozeboom mosquito collection from the Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD, has been transferred to the National Museum of Natural History, Smithsonian Institution, Washington, DC. This historically and taxonomically important collection has specimens representing 32 genera, 78 subgenera, and 763 species from most mosquito faunal areas of the world. The collection consists of 12,015 individually pinned and labeled adults, and 7,179 microscope slide preparations of immature stages and male and female genitalia. A tabulation of genera, subgenera, species, countries, faunal areas, and number of specimens for each type of preparation is provided.

Ultrastructure of infective larvae (L3) of Onchocerca volvulus (Nematoda: Filarioidea) developed in Simulium yahense in Liberia.

Third-stage infective larvae of Onchocerca volvulus were examined to elucidate the ultrastructure and the interrelations of the stoma, esophagus, intestine, and nervous system. The alimentary canal involves a cuticularized stoma with a triradiate lumen that is continuous with a similar triradiate lumen in the muscular region of the esophagus. The lumen wall may be laterally appressed or opened into a stellate form in the glandular region. Posteriad from the esophagointestinal valve, the cylindroid lumen becomes partially occluded with microvilli formed by the evaginations of the apical membranes of the intestinal epithelium. Cross sections, through this region reveal that groups of 5 radiating epithelial cells are joined near the lumen surface by junctional complexes. The alimentary canal terminates via a rectal valve and channel supported by somatic and neural cells. The central nervous system consists of a nerve ring that surrounds the muscular region of the esophagus. Related neurons support chemoreceptors and tactoreceptors of sensilla and the extensive coelomyarian and meromyarian somatic muscles. Extensive accumulations of glycogen rosettes are present in many of the muscle and hypodermal cells.

Ophthalmomyiasis interna posterior. Report of a case with optic atrophy.

PURPOSE: The prognosis for patients with ophthalmomyiasis Interna posterior is variable. In many patients the larva remains in the eye for years without inducing inflammation or loss of vision. Sometimes, however, the migrating larva involves the macula and optic nerve and results in permanent visual loss or even blindness. METHODS: Our patient presented with sudden painless loss of vision in the right eye that was caused by ophthalmomyiasis interna posterior. Hemorrhage of the optic nerve head suggested that the fly larva had entered the vitreous cavity from the optic nerve head. Subretinal tracts were sequelae of the subretinal migration of the organism. Sequential photographs documented the fly larva migration into the optic nerve head head from the vitreous cavity. The organism was removed by pars plana vitrectomy and retinectomy. RESULTS: In spite of the successful removal of the first stage fly larva, the visual acuity remained poor because of optic nerve atrophy. CONCLUSION: We suggest early removal of fly larvae to prevent damage and visual loss in cases of ocular myiasis interna posterior.

Onchocerca volvulus: parasitologic and serologic responses in experimentally infected chimpanzees and mangabey monkeys.

Six chimpanzees (Pan troglodytes) and six mangabey monkeys (Cercocebus atys) were inoculated with Onchocerca volvulus third-stage larvae (L3) of West African origin. Two chimpanzees each received 200, 300, or 400 L3, while three mangabeys each received either 50 or 250 L3. All six chimpanzees became microfilaria positive between 11 and 25 months postinoculation (PI), while two of the six mangabeys were skin-snip positive at 24 and 37 months PI, respectively. All chimpanzees developed antibodies to two native antigens of 14 and 22 kDa and to the recombinant antigens OV16, OC3.6, and OC9.3. Marked antibody responses were observed in the mangabey monkeys, and in general, the responses were similar to those observed in the chimpanzees. However, in the mangabeys, these responses did not generally manifest themselves until later in the infection. The results of this study suggest that in chimpanzees, the smallest inoculum used, 200 L3, was sufficient to initiate consistent infections that had parasitologic and immunologic parameters equivalent to animals inoculated with larger numbers of larvae. Similarly, inoculation of mangabey monkeys with small numbers of larvae appeared to be as likely to establish infection and induce immunologic responses as did inoculation of larger numbers of larvae. Microfilaria-positive chimpanzees and mangabey monkeys were examined by three conventional imaging techniques (X ray, ultrasound, and magnetic resonance imaging (MRI)), but no adult worms or nodules could be identified in any animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimates of population size, dispersal, and longevity of domestic Aedes aegypti aegypti (Diptera: Culicidae) by mark-release-recapture in the village of Shauri Moyo in eastern Kenya.

Estimates of the absolute size of a domestic population of Aedes aegypti aegypti (L.) were made in the Rabai area of Kenya, based on a single release, followed by either single or repeated recaptures. From single recapture within 24 h of release, the size of the female Ae. a. aegypti population in the Shauri Moyo village was estimated by the Lincoln index to be 365. Using a single release and repeated recaptures, population size was estimated by Jackson's positive method to be 337. Depletion of the unmarked females by daily removal sampling provided us with two additional, direct estimates of the size of the village population (451 by Kano's method and [464 +/- 18.9 SEM] by the Moran-Zippin method). The longevity of marked females was at least 9 d. The day-to-day movement of marked mosquitoes revealed the dispersal pattern of Ae. a. aegypti among houses clustered within different distance zones. Marked mosquitoes reached all houses of the village within 24 h of release. After 5 d, the dispersion of both marked and unmarked mosquitoes among houses was similar.

Aedes (Gymnometopa) mediovittatus (Diptera: Culicidae) as an experimental vector of Brugia pahangi and B. malayi (Spirurida: Filariidae).

To test the susceptibility of Aedes (Gymnometopa) mediovittatus to infection with Brugia pahangi and Brugia malayi, females originating from the suburbs of San Juan, Puerto Rico, were fed on infected gerbils (Meriones unguiculatus). On average, 39.2% of the Ae. mediovittatus females became infected with L3 larvae of B. pahangi and 47.4% with B. malayi. The average number of infective L3 larvae of B. pahangi and B. malayi dissected from mosquitoes was 2.6 +/- 1.2 and 2.9 +/- 1.0, respectively. The largest number of L3 in a single mosquito was 16. After 10 d of development in the mosquitoes, L3 larvae of both Brugian species were found in greatest number in the thorax, in lesser number in the head/proboscis, and in least number in the abdomen. Ae. mediovittatus may serve as a useful laboratory model for the study of genetic susceptibility and refractoriness of mosquito vectors to filarial parasites.

Diurnal biting activity and transmission of Onchocerca volvulus (Filariata: Onchocercidae) by Simulium yahense (Diptera: Simuliidae) in Liberia.

To determine the influence of meterological factors on the diurnal biting cycle of Simulium yahense Vajime & Dunbar (a member of the Simulium damnosum Theoblad complex), we captured host-seeking females as they landed on the exposed lower legs of humans in the Harbel area of Liberia. Biting activity was greatest during the morning hours and was characterized by a unimodal harmonic curve. Although meteorological conditions had no decisive influence on the unimodal pattern of diurnal biting activity, hourly variation in the number of S. yahense captured at human bait was affected by meterological factors (i.e., the diurnal pattern of S. yahense biting activity is regulated by an internal clock, but the number of bits during any given hour is in response to meterological conditions). The transmission of Onchocerca volvulus (Leuckart) was highest during the morning hours when high transmission potentials were in phase with peak human activity and served to maintain the hyperendemicity of onchocerciasis in the Harbel area.

Induction of protective immunity against larval Onchocerca volvulus in a mouse model.

BALB/cBYJ mice were immunized against larval Onchocerca volvulus by subcutaneous injection of normal, irradiated, or freeze-thaw-killed Onchocerca sp. larvae. The mice received challenge infections of O. volvulus third-stage larva (L3) contained in diffusion chambers implanted subcutaneously. At two-weeks postinfection, the diffusion chambers were removed and larval survival was assessed. When mice were immunized a single time with 35-krad-irradiated or normal O. volvulus L3, there was a significant reduction in the survival of challenge parasites. However, there was little or no reduction in challenge worm survival when mice were immunized a single time with freeze-thaw-killed O. volvulus L3 or fourth-stage larva (L4), or irradiated O. lienalis L3. When a second dose of freeze-thaw killed O. volvulus L3 or irradiated O. lienalis L3 was administered, there was a significant reduction in parasite survival in immunized mice. Immunization with O. volvulus L4 or a combination of L3 and L4 failed to confer protection. These results demonstrate that mice can be immunized against larval O. volvulus and that diffusion chambers are an efficient method for studying protective immunity to this parasite in a mouse model.

Cryopreservation of infective larvae of Onchocerca volvulus (Filarioidea: Onchocercidae).

Infective larvae (L3) of Onchocerca volvulus were procured in Liberia, West Africa, in the natural black fly vector, Simulium yahense. A cryobiological technique was developed to preserve L3 of O. volvulus that were fully viable after thawing. Larvae were treated before cooling with 4 cryoprotective compounds. Three compounds, dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol, were prepared with distilled water. The fourth compound was DMSO prepared in different concentrations with 0.25 M sucrose. The treatment with DMSO + 0.25 M sucrose cryoprotectant resulted in the highest survival of infective larvae. Five cooling rates between 0.5 C/min and 20.0 C/min were applied. The highest survival of L3 was with the cooling rate of 1.0 C/min. Two-step cooling of L3 was applied. In the first step, L3's were frozen to 5 levels from -10.0 C to -20.0 C, -30.0 C, -40.0 C, -60 C, and -80.0 C, and in the second step, larvae were transferred into liquid nitrogen at -196 C for rapid cooling and storage. The survival was the highest when larvae were cooled to approximately -40 C prior to transfer into liquid nitrogen. Slow, gradual, and rapid thawing procedures were applied. The survival was the highest in rapid warming.

Survival and development of larval Onchocerca volvulus in diffusion chambers implanted in primate and rodent hosts.

Third-stage larvae (L3) of Onchocerca volvulus were implanted in diffusion chambers in chimpanzees, mangabey monkeys, rhesus monkeys, squirrel monkeys, and inbred strains of mice, jirds, and rats for 3-63 days. At different times during the experimental period, larvae were recovered and assessed for their viability and development. Survival and growth rates were equal regardless of whether the implanted larvae were fresh or cryopreserved. Survival and growth rates of the larvae did not differ among the primate and rodent hosts tested, with the exception of squirrel monkeys and rats, which were resistant to infection. Molting from L3 to fourth-stage larvae began on day 3 and continued through day 14 in the primates and rodents. The primate and rodent models developed in the present study will be useful for the study of the immunology and chemotherapy of onchocerciasis.

Onchocerciasis in the forest-savannah mosaic region of Sierra Leone. Prevalence, intensity of infection, and endemicity levels.

The present study was carried out between December 1986 and January 1988. A series of 3,048 individuals (46% males and 54% females) were examined in 56 villages; onchocercal dermatitis and lymphadenitis were seen. The crude prevalence of nodules and of microfilariae amounted to 14% and 43%, respectively. The prevalence of nodules was significantly higher at the femoral trochanter than at other anatomical sites. The overall prevalence of nodules, and of microfilariae, was significantly higher in males than in females. The age-adjusted prevalence and the geometric mean microfilarial load were significantly higher in the cohort of males older than 15 years than in their female counterparts. The overall geometric mean microfilarial load was 18 microfilariae per skin snip. The correlation between prevalence and intensity of infection was poor, but was perfect between mean microfilarial density per skin snip and mean microfilarial density per milligram skin. The ratio of mean microfilarial density per skin snip to mean microfilarial density per milligram skin was 1:6. The correlation between prevalence of onchocercomata and microfilarial prevalence or microfilarial density was weak. Both of the latter criteria identified the Rokel/Seli as the river with the highest proportion of hyperendemic villages. The implications of the present findings for the epidemiology of onchocerciasis are discussed.

Duration of larval development of Simulium yahense (Diptera: Simuliidae) under natural conditions.

The duration of Simulium yahense Vajime & Dunbar larval development on a dam spillway in Harbel, Liberia, was observed to make accurate decisions regarding the frequency of larvicide treatments against this onchocerciasis vector. Larval development required a minimum of 10-12 d from eclosion to first pupation. Initial larvicidal treatment for S. yahense control would require a treatment cycle of 7 d. Once suppression of adult and larval populations is achieved, a 9-12-9-12 day treatment cycle could be adopted.

Effect of mass treatment of a human population with ivermectin on transmission of Onchocerca volvulus by Simulium yahense in Liberia, West Africa.

The impact of mass treatment with ivermectin on the intensity of Onchocerca volvulus transmission by the black fly (Simulium yahense) was evaluated on the Liberian Agricultural Company rubber plantation in Liberia, West Africa. The adult pre-treatment prevalence of onchocerciasis was greater than 80%, and the average intensity of infection was 5.35 mf/mg of skin. The drug was administered at 2 annual intervals, reaching 58-60% of the approximately 14,000 people living in 73 camps. Landing/biting catches of black flies made in central and peripheral plantation zones indicated similar fly activity before and after ivermectin treatment (man hr index of 2.1 and 2.4 within the plantation, and 10 and 10.9 outside the plantation, respectively). The number of infected flies with developing larvae (L1, L2, L3 stages) of O. volvulus in treated areas was reduced by 93.4-95%; the number of infective flies with L3 larvae was reduced by 81.7-89.3%. Parasite loads of infected (L1, L2) and infective flies (L3 stages only) outside the plantation also decreased by 86.8% and 80%, respectively. Monthly transmission potential (MTP) showed a similar decrease: from 22.9 to 5.8 (74.6% reduction) in the treated area, and from 210 to 158.8 (24.4% reduction) in untreated areas. Mass treatment with ivermectin efficiently controlled, and at least temporarily interrupted, transmission of Onchocerca volvulus by black fly vectors.

Ivermectin prophylaxis against experimental Onchocerca volvulus infection in chimpanzees.

Ivermectin was tested for possible prophylactic action against the third and fourth larval stages (L3 and L4) of Onchocerca volvulus inoculated into chimpanzees. The infective larvae (L3) were obtained from laboratory-raised black flies. Eighteen chimpanzees were inoculated, each with approximately 250 L3. Six were treated with ivermectin (200 micrograms/kg) on the day of inoculation, 6 were treated with ivermectin on day 28, and 6 were not treated. Monthly skin snips were taken for the next 30 months to detect patent infection. One of the chimpanzees treated with ivermectin on the day of infection developed a patent infection as did 4 of the 6 treated at day 28 and 4 of the 6 control animals. The results suggest that ivermectin may have a partial in vivo effect against the L3 of O. volvulus but has no effect against later larval stages of the parasite.

Large scale production of the vertebrate infective stage (L3) of Onchocerca volvulus (Filarioidea: Onchocercidae).

Laboratory-derived Simulium yahense and S. sanctipauli females were used to produce large numbers of Onchocerca volvulus infective stage larvae (L3) for use in assessing the possible chemoprophylactic potential of ivermectin in the chimpanzee model. Engorgement rates and subsequent post-prandial survival were correlated with the time at which adult flies were offered a carbohydrate source following emergence and the age of flies that fed on microfiladermic volunteers.

Population relationships of Simulium yahense and Simulium sanctipauli in the Firestone Rubber Plantation at Harbel, Liberia.

Comparative genetic differences for the phosphoglucomutase and trehalase loci were surveyed in larval and adult blackfly populations of the onchocerciasis vectors Simulium yahense Vajime and Dunbar, and S.sanctipauli Vajime and Dunbar. Genotype frequencies for each stage and all populations were at Hardy-Weinberg equilibrium, indicating that S.yahense and S.sanctipauli remain genetically distinct. S.yahense populations from three different locations were found to be genotypically comparable. The larval population of S.yahense found in closest proximity to a S.sanctipauli population was found to express the phosphoglucomutase allele 1.33, characteristic of S.sanctipauli, with significantly greater frequency than other larval populations of S.yahense. This may constitute evidence of limited genetic introgression of S.sanctipauli with the S.yahense population.

The effect of Brugia pahangi infection on survival of susceptible and refractory species of the Aedes scutellaris complex.

Life table statistics were used to examine the survival functions of filarial susceptible and refractory species of the Aedes scutellaris (Walker) group of mosquitoes, following infection with high and moderate doses of Brugia pahangi (Buckley & Edeson). Survivorship curves and hazard function curves were generated, and the median survival times and the proportions of mosquitoes surviving beyond the extrinsic incubation period of the parasite were determined. In the susceptible populations of Aedes polynesiensis Marks, Ae. pseudoscutellaris (Theobald) and Ae.tabu Ramalingam & Belkin a dose-response relationship was detected between parasite load and mortality. This relationship was characterized by a significant reduction in the proportions of infected female mosquitoes surviving at days 1 and 9 postinfection, reduction in the median survival times and an increase in the hazard rates as the infectious dose increased. The survival of the refractory species, Ae.alcasidi Huang and Ae.katherinensis Woodhill was not significantly affected by the infection. A positive correlation between microfilaraemia in the vertebrate host and parasite load in the susceptible mosquito populations was also observed. Regression analysis of the number of parasites recovered from susceptible mosquitoes at the time of death showed that mosquitoes at highest risk of dying harboured from 11.6 to 19.4 infective larvae when fed on a gerbil with sixty-five microfilariae per 20 microliters blood; this resulted in 34.4-40.2% mortality by day 9 postinfection. A mean number of 32.6-46.9 infective larvae was observed when these populations were exposed to a gerbil with a microfilaraemia of 150 mf/20 microliters and resulted in 72.8% to 80% mortality in these populations. Viable infective larvae were recovered from infected mosquitoes up to 50 days postinfection.

Quantitative studies of onchocerciasis transmission by Simulium yahense and Simulium sanctipauli in the Firestone Rubber Plantation at Harbel, Liberia.

Black fly vectors of onchocerciasis from three ecologically different Simuliid breeding habitats in the Firestone Rubber Plantation at Harbel, Liberia, were surveyed by human-biting collections conducted at weekly intervals over a 13-month period. Black flies were identified morphologically and the monthly and seasonal contribution of different vector species to the transmission of Onchocerca volvulus at each site was determined. Simulium yahense was identified as the predominant vector species at each site. Greatest populations of this species occurred during wet season (May-Oct), but its impact on transmission of onchocerciasis was most profound during dry season (Nov-Apr) when parity, infection, and infectivity were high. S. sanctipauli was the only other vector species captured, but biting populations of this species were small, and during wet season confined primarily to the vicinity of its breeding site in the Farmington River. Dry season populations of S. sanctipauli were also characterized by lower human-biting rates, and by higher rates of parity, infection, and infectivity. Monthly transmission potentials at each site were attributed primarily to S. yahense, with peak monthly transmission occurring during the dry season months of January-April. Against the WHO standard of 100 as a "tolerable" annual level of onchocerciasis transmission, annual transmission potentials for the three sampling sites were 94, 1,877, and 4,900, with highest values being calculated for S. yahense breeding sites.