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Moulting and exsheathment of the infective larvae of Onchocerca lienalis (Filarioidea) in vitro.

Fourth-stage larvae (L4) of the cattle filarial parasite Onchocerca lienalis were produced from third stage forms (L3) and maintained in vitro using a variety of culture media and feeder cell layers. Up to 50% of the L3 larvae moulted to the fourth stage in the presence of two jird (Meriones unguiculatus) cell lines, a monkey kidney cell line and also with Vero cells. Moulting took place 2-5 days after of the initiation of the cultures: These L4 parasites could be maintained in a healthy condition for up to 88 days, although no significant increases in size of these larvae were observed during the culture period. The medium giving the most promising results, both in terms of moulting and survival, was 199 supplemented with heat inactivated foetal bovine serum (20%) and glucose (2 mg/ml). The feeder layer cells could be replaced by fresh jird red blood cells. Moulting of parasites occurred in medium alone but the viability was reduced under these conditions.

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Analysis of glutathione-enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro.

Reduced glutathione (GSH), but not its oxidized form (GSSG), stimulated development of Onchocerca lienalis microfilariae to the late first-larval stage in vitro. The degree and frequency of development was dose-related with a peak of activity at 15 mM, a concentration that is similar to known intracellular levels of GSH. To determine the mode(s) of action of this multifunctional compound, other reducing agents (L-cysteine, dithiothreitol), cysteine delivery agents (N-acetyl-L-cysteine, L-thiazolidine-4-carboxylic acid, L-2-oxothiazolidine-4-carboxylic acid), cysteine analogues (S-methyl-L-cysteine, D-glucose-L-cysteine, cysteine ethyl ester), free-component amino acids of GSH (glutamic acid, cysteine, and glycine), a specific metabolic inhibitor of gamma-glutamyl synthetase (buthionine sulfoximine), and an inhibitor of gamma-glutamyl transpeptidase (gamma-glutamyl glutamic acid) were also tested at concentrations of 0.01-50 mM in this system. N-acetyl-L-cysteine at 1-5 mM and D-glucose-L-cysteine at 2.5-10 mM significantly enhanced development. In contrast to those worms maintained in GSH-supplemented medium, microfilariae exposed to GSH for only the first 24 hr showed no enhancement by day 7 in culture. Neither buthionine sulfoximine nor gamma-glutamyl glutamic acid at 0.01-35 mM inhibited the effects of 15 mM GSH or 1 mM N-acetyl-L-cysteine. Results indicate that GSH or other cysteine analogues possessing a free sulfhydryl group must be present in the extranematodal environment to support microfilarial differentiation in vitro.

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Ultrastructure of the anterior alimentary tract of infective-stage Wuchereria bancrofti (Nematoda: Filarioidea).

The anterior alimentary tract of infective-stage Wuchereria bancrofti is divided into the following segments: stoma or buccal capsule, muscular esophagus, glandular esophagus, esophageal-intestinal valve, and intestine. Invaginated external cuticle lines only the anterior stoma. External cuticle and esophageal lining are not continuous and are ultrastructurally distinct; the latter is compared morphologically to the amorphous component of elastin. The glandular esophagus is a composite structure of a stellate contractile epithelial core, surrounded by a sleeve of secretory epithelium. The glandular cytoplasm shows evidence of formation and release of dense secretory granules. At least 2 nerve cell bodies lie within the esophagus approximately 15 micrometer anterior to the esophageal-intestinal valve and their associated processes pass forward and backward through the contractile epithelium. Materials interpreted as ingested flight muscle mitochondria of the mosquito vector appear in various stages of degeneration within the intestinal lumen. It is suggested that, although simple by comparison to some other nematodes, the anterior alimentary tract of infective-stage W. bancrofti functions in the ingestion and breakdown of nutrient materials. The ultrastructure of the excretory cell likewise suggests a functional capability.

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Scanning electron microscopic study of nocturnally subperiodic Brugia malayi (Filarioidea: Onchocercidae), Narathiwat, Southern Thailand.

Scanning Electron Microscopic (SEM) observations were made on the adult females and males of nocturnally subperiodic Brugia malayi (Narathiwat, Southern Thailand) from 8-month-old intra-peritoneally infected jirds (Meriones unguiculatus). Descriptions of the morphological surfaces of anterior end, vulva, body cuticle, anus, posterior end of females and anterior end, body cuticle, cloaca, caudal papillae, spicules, sheath, posterior end of males were demonstrated. The comparison among these and other filarial parasites were also investigated.

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Acanthocheilonema sabanicolae n. sp. (Filarioidea: Onchocercidae) from the savanna armadillo (Dasypus sabanicola) in Venezuela, with comments on the genus Acanthocheilonema.

A new species of Acanthocheilonema, Acanthocheilonema sabanicolae n. sp., is described from the subcutaneous tissues of the savanna armadillo (Dasypus sabanicola) in Venezuela. The adults (females 25-30.6 mm long by 61-78 micron wide; males 10.9-15.3 mm long by 42-44 micron wide) and the microfilariae (112-131 micron long by 3-5 micron wide) are the smallest within the genus. The microfilaria, a skin dweller, assumes a characteristic C-shaped curve, when fixed in 2% formalin, which serves to distinguish A. sabanicolae from others in the genus. Moreover, the spicules are notably smaller than any others in the Acanthocheilonema. Acanthocheilonema sabanicolae is the first species in this genus to be described from edentates, and, along with A. reconditum of dogs and A. pricei of opossums, is the third species of Acanthocheilonema to be reported from South America. Morphological features, vectors, and vertebrate hosts of the genus Acanthocheilonema are reviewed.

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Studies on Dipetalonema viteae (Filarioidea) 3. Antibody-dependent cell-mediated destruction of microfilariae in vivo.

Antibody-dependent cell-mediated destruction of Dipetalonema viteae microfilariae could be demonstrated in the golden hamster using a micropore chamber technique. Microfilariae were eliminated within 24 hours in chambers of 3.0 and 5.0 microm pore size when implanted into amicrofilaremic hamsters (week 30 post infection). At peak microfilaremia (week 12 post infection), only some hamsters could efficiently destroy microfilariae. In chambers with 0.3 microm pore size, microfilariae survived for more than 3 weeks in all hamsters. In uninfected hamsters, microfilariae could only be eliminated if they had been preincubated with serum or its 19S fraction containing antibodies to the cuticle of microfilariae. The opsonizing activity of the serum was abolished by 2-mercaptoethanol treatment. The composition of cells adhering to microfilariae was always significantly different from the composition of cells which migrated into a chamber. The adhesion patterns on individual microfilariae indicated that no single effector cell type was responsible for the destruction of microfilariae. The eosinophil was the predominant cell type but neutrophils, lymphocytes and monocytes also adhered to the microfilariae. Cellular adhesion led to the immobilization of microfilariae and subsequently to their disintegration within large cell clusters. During the final stages of destruction the contribution of the monocyte became more pronounced.

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Ultrastructure of the rectum of infective-stage Wuchereria bancrofti (Nematoda: Filarioidea).

The authors have examined the ultrastructure of the rectum of infective-stage Wuchereria bancrofti by transmission electron microscopy. Our observations show that the rectum is divided into anterior and posterior segments. The cells of the anterior rectum appear to be derived from the microfilarial R (rectal) cells described by other authors. In both stages, these cells show voluminous nuclei, abundant mitochondria, and small cytoplasmic processes which contain fibrillar components. Amorphous material associated with these processes appears throughout the larval rectum and may protrude from the anus as the rectal plug. In the specimens examined, a patent lumen could not be traced completely through the anterior rectum. The posterior rectum has no counterpart in published accounts of microfilarial ultrastructure and probably arises during larval morphogenesis; it is lined with invaginated body cuticle, overlaid by a single layer of epithelial cells which may be of hypodermal origin.

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Studies on the Onchocerca (nematoda: Filarioidea) found in cattle in the United States. I. Systematics of O. gutturosa and O. lienalis with a description of O. stilesi sp. n.

Onchocerca gutturosa and O. lienalis are redescribed and comparative morphological descriptions provided. In addition, a third species of Onchocerca is described from cattle in the United States. This third species, Onchocerca stilesi sp. n., localizes in the connective tissues around the stifle joint, and can be distinguished morphologically from O. gutterosa and O. lienalis. Onchocerca gutturosa and O. lienalis apparently have widespread distribution in the eastern United States; however O. stilesi has been reported only from Minnesota.

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Dipetalonema (Acanthocheilonema) didelphis sp. n. (Nematoda: Filarioidea) from opossums, with a redescription of D. (A.) pricei (Vaz and Pereira 1934).

Dipetalonema (Acanthocheilonema) didelphis sp. n. recovered from the subcutaneous and periesophageal connective tissues of Didelphis virginiana from several localities in the United States and of D. marsupialis from Colombia is described. Dipetalonema didelphis sp. n. resembles D reconditum and D. mansonbahri in size, in lacking a two-segmented buccal capsule, and in the female having a long, digitiform tail. Dipetalonema reconditum, however, has a much longer glandular esophagus and D. mansonbahri has a rounded cephalic extremity delimited by a necklike region. The male of D. didelphis sp. n. differs from both in the structural details of the spicules and in having a higher spicular ratio. The microfilaria is much shorter than that of either D. reconditum or D. mansonbahri. Dipetalonema (A.) pricei adults are redescribed from the paratype specimens, and the microfilaria is also described. Dipetalonema pricei most closely resembles D. dracunculoides in size and in the presence of a well-developed, two-segmented buccal capsule, but the latter species has a much longer glandular esophagus. These two species also differ in the appearance of the spicules.

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Studies on Dipetalonema viteae (Filarioidea). 4. Passive transfer of immunity to circulating microfilariae by spleen cells.

Passive transfer experiments provided clear evidence for a role of spleen cells in the immunity of hamsters against Dipetalonema viteae microfilariae. 106 or more spleen cells from postpatent (amicrofilaraemic) hamsters suppressed incipient microfilaraemia in syngeneic recipients when transferred at either week 2 or week 6 of a primary infection. Spleen cells from microfilaraemic donors (week 15-16 post infection) were also capable of transferring immunity against microfilariae. The spleen cell transfer never affected the survival of adult worms. After transfer of spleen cells from either patent or postpatent hamsters, antibodies to the cuticle of microfilariae could be found in the sera of recipients as early as week 8 of a primary infection.

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Dipetalonema viteae (Filarioidea): development of the infective larvae in vitro.

The development of Dipetalonema viteae third stage larvae was attempted in vitro. A monophasic culture system consisting of BHK-21 medium supplemented with 10% tryptose-phosphate broth and 15% fresh jird serum allowed the growth of previously in vivo triggered larvae, but not of those isolated from ticks. The larvae could complete their third moult and grew on a fourth stage larvae up to 5 mm. This development was comparable to that observed in vivo. The presence of an irradiated hamster kidney cell feeder layer could replace an in vivo trigger. The development of the infective larvae took place up to the third moult. Some larvae completed the moult to the fourth stage and reached a maximum length of 3 mm after 14 days. These culture systems offer the possibility to study in vitro the complete development of the infective larvae to fourth stage larvae.

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