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An improved method of examining adult Onchocerca volvulus worms.

Adult Onchocerca volvulus worms were extracted from alcohol-fixed nodules by collagenese digestion. The worms were stained in Mayer's haematoxylin and brought up into glycerol. In this viscous fluid the stained and cleared worms could be unravelled in sequential lengths of 4-6 cm and mounted in glycerol on slides for longitudinal examination of their structure. The worm lengths were then cut into small pieces, 0.5 cm long, which were implanted sequentially into slits in a brick-shaped block of formalin-fixed calf's brain (white matter) also in glycerol. The brain blocks were then processed in paraffin wax and cut as 6 microns histological sections, enabling transverse sections of the worm, stained as required, to be examined in sequential order.

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[Vector capacities of Similium damsnosum s.l and risk for Onchocerca volvulus transmission in Inga (Democratic Republic of Congo)].

This article analyzes the results of a one-year entomological survey conducted near the Yalala rapids in the Inga region, Democratic Republic of Congo, prior to the implementation of Community-Directed Treatment with Ivermectin (CDTI). A vector control program had been undertaken from October 1969 to December 1980, 20 kilometers upstream from the study site. The results of the present study show that during 122 collection days, 5,824 females were captured, of which 3,899 were dissected; 2,001 (51.3%) were parous while 44 (2.2%) were infected while 4 (0.2%) were infective. The crude Annual Transmission Potential (ATP) was 65 infective larvae per man and per year at the river as against 12 in Yalala village located two kilometers from the rapids. The average biting rate was 5 flies/man/day during the dry season and 90 during the rainy season. The vector capacity of similium shows that the risk of transmission of Onchocerca volvulus to the human population was not only reduced in time (2-3 months), but was also negligible all year round.

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Survival of Onchocerca volvulus in nodules implanted in immunodeficient rodents.

Onchocerca volvulus is an obligate human parasite, and its study has been difficult due to an inability to maintain it outside the human host. We report the successful transplantation of onchocercomata containing living adult O. volvulus worms into immunodeficient C.B.-17.scid/scid (scid) mice or athymic rnu/rnu (nude) rats. Living, motile worms containing viable microfilariae were present in onchocercomata recovered from scid mice or nude rats for up to 20 wk, establishing a novel animal model for future investigation of O. volvulus.

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Suramin and the time it takes to kill Onchocerca volvulus.

The paper records the numbers of 1-2 mm shotty papules developing in the author's skin after 2-day courses of diethylcarbamazine (DEC) repeated every 16 days before, during and after a 7.1 G suramin course for cutaneous onchocerciasis. Assuming, from biopsy evidence, that each papule represented a dead Onchocerca volvulus microfilaria (mf), the number of mfs reaching the skin every 16 days did not begin to fall until 96 days after the first full dose of suramin; and only reached zero by day 224. The histopathology of nodules excised from Cameroonian patients before, and at intervals of 56-335 days after, the start of a 7.1 G suramin course, revealed changes in the worms that correlated over time with the disappearance of mfs from the skin. Suramin sterilized and killed the male worms between days 77 and 105 and, in the females, it adversely affected the staining and subsequent development of small morulae from about day 56. It was estimated that new embryogenesis ceased about 56-77 days after the first full dose; development of the last viable embryos to mfs was complete by about 136 days; and the last mfs, perhaps having reduced vitality, emerged from the females by 160 days and reached the skin within 16-32 days.

Adult↗

Onchocerca volvulus: in vitro killing of microfilaria by neutrophils and eosinophils from experimentally infected chimpanzees.

Peripheral blood leukocytes from chimpanzees experimentally infected with third stage larvae, or with nodular microfilaria (mf) of Onchocerca volvulus, were evaluated for their ability to kill nodular mf in vitro. Inoculation of chimpanzees with third stage larvae, and prospective follow up, permitted each animal to be classified as patent, infected but not patent, or post-patent. Neither lymphocytes nor monocytes significantly adhered to, immobilized, or killed mf. However, neutrophils or eosinophils killed mf effectively in the presence of immune serum. Neutrophil and eosinophil enriched cell preparations from control animals were able to kill mf with serum from infected animals, but to a significantly lesser degree than when both cells and serum were taken from infected animals. Chimpanzees inoculated with mf showed a progressive increase in cellular killing of mf after a second inoculation which surpassed that seen with animals inoculated with third stage larvae. Antibody-mediated killing of mf by neutrophils and eosinophils may be important in the termination of patency in this infection.

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The embryogenesis of Onchocerca volvulus over the first year after a single dose of ivermectin.

Adult Onchocerca volvulus, extracted from nodules before, and at intervals of two weeks to 12 months after, a single 150 micrograms/kg dose of ivermectin, were examined longitudinally and by sequential transverse sections. The mean number of male worms per nodule fell, and the proportion of nodules with no male worm rose, within two weeks of ivermectin and remained so for 12 months. In female worms, at intervals after ivermectin, the percentages of the length of the lower genital tracts occupied by embryos at each stage of development, or by degenerating ova, embryos and microfilariae (mfs), were recorded: (a) in un(re-)inseminated worms whose original embryogenesis was continuing and in those in which it was completed; and (b) in worms, reinseminated post-ivermectin, in which a new embryogenesis had begun. The results indicated that: (a) the time needed for the zygotes of O. volvulus to develop to mfs is 8-12 weeks; (b) nearly 40 percent of females had not resumed mf production by 12 months after treatment; (c) many intrauterine mfs had not degenerated within the first two weeks of ivermectin; (d) some of the last embryos to mature to mfs did not degenerate but accumulated temporarily in the anterior uteri 8-16 weeks after ivermectin.

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Surface area calculation for Onchocerca volvulus microfilariae.

The pathogenesis of tissue damage in filarial infections involves the generation of inflammatory mediators by interaction of parasite surfaces with the host immune response. In the presence of a heavy parasite burden, the tremendous potential for development of adverse local or systemic inflammatory responses may be appreciated by knowledge of the cumulative surface area represented by circulating microfilariae. The approximate surface area of a single microfilariae of Onchocerca volvulus was calculated using mathematical formulae for standard geometrical shapes and values derived from serial measurements of electron micrographs. Numerical values for the surface area represented by large numbers of microfilariae are given and compared to surface areas of the host which can harbor many millions of microfilariae.

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Experimental ocular onchocerciasis in cynomolgus monkeys. IV. Chorioretinitis elicited by Onchocerca volvulus microfilariae.

Onchocerciasis is a major cause of blindness worldwide, and much of the blindness is caused by onchocercal chorioretinitis. In an experimental animal model for ocular onchocerciasis, intravitreal injections of 10,000 live Onchocerca volvulus microfilariae isolated from infected humans into the eyes of cynomolgus monkeys (Macaca fascicularis) resulted in patchy, progressive loss of retinal pigment with pigment clumping. Areas of pigment loss were less extensive in animals that had been sensitized with microfilariae. Intravitreal injections of dead O. volvulus microfilariae resulted in mild vitritis with relatively less clinical change noted in the retina and choroid. Histopathologic examination revealed thinning and loss of outer retinal layers with pigment migration into the retina, and inflammation was more pronounced in eyes that received live microfilariae. Clinical changes appeared in eyes receiving live microfilariae before the development of significant antibody or cell-mediated immune responses. O. volvulus microfilariae appear to be more suitable than O. lienalis microfilariae in producing lesions which resemble human onchocerciasis in the primate model.

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Isolation of living adult Onchocerca volvulus from nodules.

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.

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Guatemalan human onchocerciasis. II. Evidence for IgG3 involvement in acquired immunity to Onchocerca volvulus and identification of possible immune-associated antigens.

Ag-specific isotypic differences in immune response to Onchocerca volvulus Ag were assessed for 778 long term residents of endemic Guatemalan areas by quantitative ELISA with 5-min incubation steps and immunoblot. The study population was separated into five groups based on clinical status: N+F+, N+F-, N-F+, N-F-H+, and N-F-H-, where N = O. volvulus adults (nodule), F = microfiladermia, and H = history of O. volvulus infection. A subset of 44 individuals with high exposure to onchocerciasis from the N-F-H- group were critically evaluated and designated as "putatively immune." IgG1 reactivity to O. volvulus Ag was elevated in the majority of infected persons, but not in putatively immune individuals. Specific IgG3 levels, however, were equally elevated in all groups. The majority of N+F- persons also had elevated IgG1 levels, but they were lower than those found in F+ persons. IgG3 reactivities to a group of antigens at 20 kDa (GP20) were seen in many uninfected persons and some N+F- persons. In contrast, most F+ persons, react to this Ag with IgG1 and not IgG3. A mangabey inoculated with the infectious larval stage of O. volvulus (L3), but showed no signs of infection, began to recognize GP20 at 2 wk postinoculation. Early recognition of GP20 was possibly elicited by the larval stage. Purified nodule Ag from N+F+ individuals contained GP20, however, identical nodule Ag prepared from N+F- individuals did not. These data suggest that GP20 Ag may be common to both uterine microfilaria and the infectious larval stages. The fact that GP20 is predominantly recognized by IgG3 in putatively immune persons and some N+F- persons suggests that this increased IgG3 activity may be important in acquired immunity to onchocerciasis.

Adolescent↗

Experimental onchocerciasis in chimpanzees. Cell-mediated immune responses, and production and effects of IL-1 and IL-2 with Onchocerca volvulus infection.

Nine of eighteen chimpanzees inoculated with infective third-stage larvae of Onchocerca volvulus developed patent infection with microfilariae in skin biopsies. In all infected chimpanzees the in vitro cellular reactivity to O. volvulus adult worm-derived Ag (OvAg) increased significantly after exposure to third-stage larvae. However, during prepatency the in vitro cellular responses to OvAg decreased gradually in subsequently mf positive (patent) animals, and returned with patency to values not different to those before infection. In non-patent chimpanzees cellular responses remained significantly higher than before infection. Stimulation of PBMC in vitro with bacterial Ag and mitogen did not show any differences between the experimental groups through 20 months p.i. The addition of exogenous IL-2 did not restore the impaired responses of PBMC to OvAg in patent animals. Exogenous IL-2 elicited an additive increase of the cellular response to OvAg in nonpatent, and a mitogenic effect to OvAg in patent animals. Selective depletion of adherent, suppressor/cytotoxic (CD8+), NK cells (CD16+) and the use of autologous serum had no effect on antigenic and mitogenic cellular responsiveness. OvAg-induced IL-2 production decreased after patency, whereas, IL-1 production was significantly greater in both patent and nonpatent than in control chimpanzees. In summary, these data demonstrate that experimental O. volvulus infection in chimpanzees stimulated a substantial cell-mediated immune response. In patent chimpanzees an OvAg-specific cellular hyporesponsiveness occurred before onset of patency, possibly due to decreased IL-2 production and responsiveness.

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The use of monoclonal antibody-based ELISAs to monitor chemotherapeutic effects in the bovine-Onchocerca gibsoni drug screen.

Three monoclonal antibodies directed towards antigens of Onchocerca gibsoni were used in antigen detection ELISAs to detect parasite antigens in sera from 100 cattle infected with O. gibsoni, in trials with the filaricidal compounds CGP 6140, CGP 20309, CGP 20376, CGP 21833, CGP 24589 and CGP 26702. Measurable levels of parasite antigens were highly variable, both within and between treatment and control groups of animals, with no consistent trends which related to time after treatment, micro or macrofilaricidal effects against O. gibsoni, or dose rate for any of the compounds used. It was concluded that these assays were unsuitable as a method of identifying drug-induced damage to O. gibsoni following the administration of these compounds. A detailed protocol for selecting suitable assays is discussed.

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The use of monoclonal antibody-based ELISAs to monitor the efficacy of drugs against male Onchocerca gibsoni in vitro.

Four monoclonal antibodies directed against antigens of Onchocerca gibsoni were used in antigen detection ELISAs to monitor the efficacy of CGP 20309, CGP 20376, CGP 21833, CGP 24589 and CGP 26702 at 5 micrograms/ml against male O. gibsoni in vitro. No significant differences (P less than 0.05) in antigen output between treated and control groups of parasites were recorded. However, consistently higher levels of antigen from treated (CGP 21833) as compared to control parasites were measured with all four assays, with differences being higher in the first 2 to 3 days post treatment than subsequently. The sensitivity of comparisons between groups was reduced by the high variability in output of antigen both between worms and also from the same worm, in part as a result of mechanical damage to worms sustained during collection or manipulation in vitro. This problem was reduced by zero handling once worms were established in vitro and it is recommended that future work should include a 24 to 48 hour period before treatment commences to detect raised antigen levels associated with physically damaged parasites so they can be excluded. It was concluded that this type of assay has no intrinsic technical or logistical advantage over other published methods of assessing drug-related damage in in vitro filarial screens. Nevertheless, further work using antigen detection ELISAs in this context is justified since these assays, unlike all other methods of assessing drug-induced damage in vitro, have direct application for use in identifying chemotherapeutic effects against similar parasites in vivo.

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Migration and death of skin-dwelling Onchocerca volvulus microfilariae after treatment with ivermectin.

The effects of a single dose of ivermectin (122-200 micrograms/kg) on Onchocerca volvulus microfilariae (mf) in skin and lymph nodes were studied histologically in a qualitative and quantitative manner over seven days after treatment. Ivermectin caused mf to move from the subepidermal layer into the deeper layers of the dermis, subcutaneous fat and connective tissue, and the regional lymph nodes. No mf were seen migrating upwards through the epidermis. Dead mf were not seen before 24 hr and the numbers found dead in the dermis were far fewer than the total numbers in the skin before treatment. Inflammatory cellular reaction around dead mf in the tissues was usually minimal. The results suggest that most of the mf which disappear from the skin may be destroyed in the lymph nodes and that the histological reaction excited by mf dying under ivermectin is less violent than that after diethylcarbamazine.

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The effects of ivermectin on the viability of Onchocerca lienalis microfilariae in vitro and on their subsequent development in the blackfly vector, Simulium ornatum.

Experiments examined the sensitivity of Onchocerca lienalis microfilariae (mf) to ivermectin in vitro using a range of parameters to measure viability. Incubation in drug (1 x 10(-4) and 5.71 x 10(-8) M) caused an immediate decrease in motility levels in a concentration dependent fashion with a continuing decline over 24 h; the inclusion of a monkey kidney cell (LLCMK2) feeder layer in the culture system partially abrogated drug effects. The use of feeder cells allowed the longer-term culture of mf; incubation in drug at low concentrations (5.71 x 10(-8) and 1 x 10(-10) M) significantly reduced motility levels during a 17 day trial. Using MTT colorimetry it was found that 10,000 untreated mf per replicate sample were required to produce a sufficient quantity of formazan to make comparisons, and that formazan formation was most rapid during the first 30 min of incubation in MTT but continued up to 3-4 h. Following incubation of mf in ivermectin (1 x 10(-4) and 5.71 x 10(-8) M) for 24 h, only the higher concentration inhibited formazan formation (51%), while motility levels were reduced by 94% and 45% respectively. Using the same drug concentrations, mf viability was assessed by measuring the uptake of [3H]2-deoxy-D-glucose; uptake was only significantly reduced (40%) by the higher concentration, while motility levels were reduced by 77% and 63% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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On the reproductive activity of the female Onchocerca volvulus.

Onchocerca volvulus worms, extracted from nodules by collagenase digestion, stained with haematoxylin and cleared in glycerol, were unravelled for longitudinal examination and later embedded in brain blocks for study of serial transverse sections. A classification system for female worms is proposed, based on the reproductive status of 446 worms from Guatemala, 94 from Liberia and 125 from Mali. They were categorized into fecund, inseminated specimens; uninseminated, but potentially fertile specimens, shedding ova destined to degenerate; worms changing from the uninseminated to the inseminated state and vice versa, which were few in number; old worms, with degenerate ovaries, whose genital tracts were either empty or had disappeared; and moribund or dead worms, characterized by loss of turgor, collapse and degeneration, calcification, or invasion by polymorphic, basophilic cells. Potentially fertile worms shed oocytes continuously and, when they were inseminated, embryonic development ensured. No evidence was found of a periodic cycle of reproduction. Inseminated worms were found in nodules without a male worm, and uninseminated worms in nodules harbouring male worms. Measurements are recorded of portions of the female reproductive tract and of the length of uterus occupied by the various embryonic stages in fully fecund worms. A significant difference in the length of the body behind the first and second ovaries was observed as between worms from West African savanna (Mali) and forest (Liberia). Limited observations were also made on meiosis in the oocyte, penetration of the oocyte by sperm, formation of the ovum, syngamy and zygote formation.

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Morphological alterations of male Onchocerca volvulus after in vitro exposure to mel w and milbemycin a confirming the results of viability tests.

The suitability of two viability parameters used for screening of antifilarial activities of new compounds was examined by parallel observation of the morphology. Male Onchocerca volvulus were exposed in vitro to 10 mumol mel w and milbemycin a and examined by light and transmission electron microscopy. The viability was assessed by measurement of the motility, using a micromotility meter and by determination of tetrazolium reduction. Already twelve hours after exposure to mel w the muscles of the body wall showed severe damage. After 36 hours the other tissues revealed degenerative changes and after 60 hours disintegration of all tissues was observed. Effects on the morphology caused by milbemycin a were seen earliest after 60 hours. Condensed cytoplasm in the hypodermal layer and beginning degeneration of spermatogenic stages indicated drug activity. The time-point of appearance of these drug induced morphological alterations was in accordance with the decrease of the motility indices and the degree of tetrazolium reduction. Morphological alterations indicating irreversible damage of worm tissues are a reliable parameter to detect macrofilaricidal activity. The good agreement between the results of the morphological examination and the assessment of the motility and the tetrazolium reduction confirms the suitability of the latter two assays for in vitro drug screening with O. volvulus.

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Antigenic and dynamic properties of the surface of Onchocerca microfilariae.

We analyzed the antigenicity and stability of the surface of skin microfilariae (mf) of Onchocerca cervicalis, a horse parasite. These mf express antigens on their surface that are cross-reactive with the cattle parasite O. lienalis and with the human parasite O. volvulus. The surface of living O. cervicalis mf was radioiodinated using Iodogen and the labeled components were solubilized in buffers containing sodium dodecyl sulphate (SDS), or extracted with the milder detergent octyl-beta-D-glucopyranoside (OGP). Electrophoresis of this material showed seven prominent bands, one of which (14 kDa) was specifically precipitated by antisera from rabbits immunized with mf from either O. cervicalis, O. lienalis, or O. volvulus, and by human sera obtained from infected individuals in Chiapas, Mexico. Other components were precipitated by either the rabbit or the human sera. In addition, antisera from mice immunized with O. cervicalis mf bound specifically to the surface of freeze-thawed uterine O. lienalis and O. volvulus mf as detected by immunofluorescence. This fluorescence was lost from the surface of O. cervicalis mf in a temperature-dependent fashion. Live mf incubated on ice with mouse anti-mf antisera and secondary FITC-GAM, showed uniform surface fluorescence. When these mf were incubated at 37 degrees C, but not at 0 degrees C, the fluorescent pattern changed with time. First, small non-fluorescent patches arose, followed by an increasingly wide belt devoid of fluorescence, and finally, no visible fluorescence. These changes in the mf surface suggest potential mechanisms for immune evasion by filarial parasites.

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