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.     

The detoxification of xenobiotic compounds by Onchocerca gutturosa (Nematoda: Filarioidea)

In common with other helminths O. gutturosa appears to lack cytochrome P450 linked phase 1 enzymes and so its ability to metabolize aromatic nuclei may be severely restricted. The parasite could reduce azo- but not nitro-compounds and low levels of epoxide hydrolase activity were also detected. Glutathione S-transferase was the only phase 2 enzyme which could be demonstrated in O. gutturosa. High levels of glyoxalase I and in particular glyoxalase II were found in the parasite, suggesting an important role for these enzymes in detoxification.     

Onchocerca lienalis: comparison of techniques for the cryopreservation of microfilariae within skin-snips or free of host tissues.

Onchocera 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 cryopreserved 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.     

An in vitro radiolabel uptake viability assay for Onchocerca microfilariae

A radiolabel uptake viability assay for Onchocerca cervicalis using [3H]2-deoxy-D-glucose in Hanks' balanced salt solution, pH 7.5, at 30 C is described and compared to the traditional visual motility assay. A correlation of r = 0.92 between the assays was found, with the radiolabel uptake method apparently a more sensitive indicator of microfilarial viability.     

Studies of the growth-regulating effects of ivermectin on larval Onchocerca lienalis in vitro

At concentrations of 0.1-100 ng/ml ivermectin inhibited L3-L4 molting by Onchocerca lienalis in vitro. The degree of inhibition was dose-dependent with a significant effect apparent at 0.1 ng/ml and complete inhibition occurring at 100 ng/ml. The ED50 for molt inhibition was 0.19 ng/ml. Molt-inhibiting levels of the drug were not acutely toxic to the worms. In the presence of 10 ng/ml, a concentration giving 95% molt inhibition, motility at day 7 postinoculation was 71% of that seen in nontreated controls. A more pronounced effect on motility was apparent in larvae under long-term cultivation in the presence of ivermectin. Kinetic studies indicated that the majority of the larvae respond irreversibly to the drug within the first 2 hr of exposure. Twenty-four hours of exposure were required for a maximal response. The inhibitory effects of ivermectin were less pronounced if larvae were allowed to develop under normal culture conditions for 24 or more hours prior to the initiation of drug treatment.     

Onchocerca sweetae (Nematoda: Filarioidea): notes on the intermediate host.

Microfilariae of Onchocerca sweetae are broadly distributed in the superficial layers of the dermis of the water buffalo (Bubalus bubalis). A total of 2855 insects representing 20 species were collected from O. sweetae-infected bait buffaloes. Only one species, Culicoides sp. "M", ingested microfilariae from buffalo skin. Larval development of O. sweetae was observed in the thorax of this species. Atotal of 829 insects, representing 7 species and including 749 parous Culicoides spp. were collected from light and Manitoba traps. Developing filarioid larvae were observed only in Culicoides sp. "M". It is concluded that Culicoides sp. "M" is a natural intermediate host of O. sweetae in the Northern Territory of Australia.     

Immunity to Onchocerca lienalis microfilariae in mice. II. Effects of sensitization with a range of heterologous species

The model of Onchocerca lienalis microfilariae (mff) injected into CBA and T.O. strains of mice has been used to examine immunity to skin-dwelling microfilariae following exposure to a range of species of helminths. Mice which had received a primary infection with O. cervicalis mff were significantly resistant to challenge with O. lienalis mff (58% reduction relative to challenge controls). Immunization with the uterine contents (eggs and mff) of O. lienalis, O. gutturosa or O. volvulus conferred equivalent levels of protection against challenge with O. lienalis mff (66 to 75%). Similar results were obtained with immunizations in mice that employed either fresh or freeze-killed eggs of O. gutturosa. Significant reductions in the recoveries of O. lienalis mff were also demonstrated following the intraperitoneal implantation of adult male worms of O. gutturosa (30 to 52%), the adults of either sex of Dipetalonema viteae (60%), or after infection with Trichinella spiralis (27 to 81%). Infections with the trematode, Schistosoma mansoni, had a negligible effect on mff recoveries. It is concluded that partial resistance in mice to Onchocerca mff may be stimulated by factors, yet to be determined, that are neither stage nor species-specific.     

In vitro haemagglutination and attenuation of microfilarial motility by haemolymph from individual blackflies (Simulium ornatum) infected with Onchocerca lienalis

Two in vitro tests were used to investigate the effect of Onchocerca lienalis Stiles infection on the haemolymph of Simulium ornatum Meigen. The first of these examined the effect of infected haemolymph on the motility of fresh O. lienalis or Brugia pahangi Buckley & Edeson microfilariae. Incubation of haemolymph from individual flies with fresh microfilariae was performed in the wells of Terasaki micro-tissue culture plates. Motility of both species of parasite was found to be significantly attenuated when compared to worms incubated in control haemolymph groups. The second assay was that of agglutination of cat erythrocytes in the presence of haemolymph from individual flies, also performed in Terasaki plates. This test demonstrated significant increases in the rates of haemagglutination in the haemolymph of O. lienalis infected blackflies. The titre appeared to increase during the initial 5 days of infection up to a level of 1/32+, but then fell between day 5 and 7 to a maximum level of 1/2. The proportion of flies exhibiting haemagglutination also rose following infection. Despite the apparent absence of melanization and encapsulation, simuliids may have at least two humoral haemolymph components available to them for parasite regulation; a fast-acting factor responsible for rapid parasite death, and more specific agglutinins, possibly lectins. The role of the latter in defence is as yet unclear.     

Surface carbohydrate changes on Onchocerca lienalis larvae as they develop from microfilariae to the infective third-stage in Simulium ornatum

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.     

A new species of Pelecitus (Filarioidea: Onchocercidae) from the endangered Tehuantepec jackrabbit Lepus flavigularis

Pelecitus meridionaleporinus n. sp. from the Tehuantepec jackrabbit is described. The new species differs from Pelecitus helicinus (Molin, 1860) in having delicate transverse striations, a salient vulva, and a readily apparent preesophageal ring; P. helicinus has teardrop cells around the vulva, which are lacking in the species presently described. The new species is different from Pelecitus scapiceps (Leidy, 1886) in having the vulva anterior to the esophageal-intestinal junction and wider lateral alae. Pelecitus scapiceps is found in the tarsal bursa of the hind feet of lagomorphs, whereas P. helicinus is found around tendons of legs and feet of birds. Pelecitus meridionaleporinus n. sp. occurs in the subcutaneous tissue at the base of both ears. This is the second species in Pelecitus Railliet and Henry, 1910 that occurs in New World lagomorphs, and the third found infecting mammals.     

Chromosomes of Onchocerca volvulus (Spirurida: Onchocercidae): a comparative study between Nigeria and Guatemala

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

A new species of Dipetalonema (Filarioidea: Onchocercidae) from Ateles chamek from the Beni of Bolivia

We describe a new species of Dipetalonema occurring in the body cavity of Ateles chamek (Humboldt, 1812) from north-central Bolivia. Morphologic characters serving to separate Dipetalonema yatesi n. sp. from known forms include a vagina vera with a simple tube and thin walls and a left spicule, which possesses a handle shorter than the lamina (ratio 2.7); the latter displays an anterior membranous alae similar in length to the terminal flagellum, a distal extremity of the left spicule within a simple hook and a membrane, phasmids at the basis of the lappets, and heterogeneous muscles occupying the whole cavity. Dipetalonema yatesi n. sp. can be separated from Dipetalonema robini, Dipetalonema gracile, and Dipetalonema graciliformis, between other characters, in having a simple vagina vera instead of a sinuous one, and from Dipetalonema caudispina and Dipetalonema freitasi in having the lamina of the left spicule divided in a membranous alae and a terminal flagellum.     

Lemdana latifi n. sp. (Filarioidea: Onchocercidae) from the red jungle fowl (Gallus gallus spadiceus)

Lemdana latifi n. sp. was found in connective tissues around the trachea and crop and in the body-cavity of seven of 14 Malayan red jungle fowl Gallus gallus spadiceus. The new species is described and illustrated. Morphologically it is most closely related to Lemdana pavonica and Lemdana francolini. Lemdana latifi is distinguished from the eight valid species of Lemdana by the mean spicular ratio of 1.7:1; the right spicule with a right margin 18–29% (15–31 m; mean 24 m) longer than the left margin; the distal half of the left spicule twisted and S-shaped; and the absence of unpaired papillae at tip of male tail. The new species has smaller adults, a shorter left spicule and a shorter glandular oesophagus than those of L. pavonica and a wider male, shorter spicules and a longer muscular oesophagus than those of L. francolini. The male of L. latifi is 7–9 (8.1)mm long, the left spicule 164–215 (184)m long and the right spicule 98–117 (108)m long. The female is 17–23 (21)mm in length. Sheathed microfilariae from blood smears are 78–100 m long and those from the uterus are 89–103 m long. This is the sixth valid species of Lemdana in the Phasianidae.     

Experiments in vitro with Litomosoides carinii (Nematoda: Filarioidea). I. Maintenance of adult females and microfilariae as well as release of microfilariae in different culture media (author's transl)

Embryos of L. carinii continue intrauterine development to microfilariae and are totally released into the medium within 5--6 days when the latter (Tc 199) is changed daily and air is used as the gas phase. Oogenesis or further fertilization of eggs, however, does not occur in vitro in any of the media examined by us. One female releases 140 X 10(3) microfilaria/day on an average in vitro within 5--6 days. Mean initial numbers of 300 X 10(3) Mf/female/day are observed. Addition of equine serum inhibits microfilarial release in vitro; normal cotton rat serum prolongs survival of females while total numbers of released microfilariae or retained embryonic stages are not increased. The serum of post-patent animals does not influence the numbers of released microfilariae or their viability or survival of females. Microfilariae released in vitro in Tc 199 + 33% normal cotton rat serum survive for more than 8 days, when air is used as the gas phase and the medium is changed daily. Microfilariae isolated from the blood of patent animals survive for at most 6 days, at a 48-hourly change of medium survival does not even exceed 4 days.