Characterization of the heartworm Acanthocheilonema spirocauda (Leidy, 1858) Anderson, 1992 (Nematoda: Onchocercidae) in Scandinavia.

The heartworm Acanthocheilonema spirocauda (Leidy, Proc Acad Nat Sci Philadelphia 10:110-112, 1858) Anderson, 1992 is described from material collected from harbour seals in Scandinavia and compared with types and other specimens described by Anderson (Can J Zool 37:481-493, 1959) from harbour seals in eastern USA. Most morphometric characters of the material from USA fall within the ranges established for the Scandinavian one. Some intraspecific variability in the organisation of papillae on the male tail was detected among the Scandinavian specimens. Differences between the specimens from Scandinavia and Eastern USA are also found in the organisation of papillae on the tail of males and females. An excretory pore was not discernible, but a clearly hemizonid-like structure is described. For the first time, scanning electron micrographs present external morphological structures of the species.

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.

Trans-species transfer of Wolbachia: microinjection of Wolbachia from litomosoides sigmodontis into Acanthocheilonema viteae.

Intracellular bacteria of the genus Wolbachia are found in most filarial nematodes, but are lacking in some species like Acanthocheilonema viteae. Due to their symbiotic nature and their role in the pathology of filarial infections they are considered to be potential targets for intervention against filarial infections in man. Infection of A. viteae (a species which does not naturally carry Wolbachia) with Wolbachia bacteria could allow comparative studies on the effect of the endobacterium on the parasite and on the host's immune systems. As a step towards such studies we microinjected adult female A. viteae with Wolbachia obtained from Litomosoides sigmodontis. The bacteria were isolated from L. sigmodontis by density-gradient centrifugation, microinjected into A. viteae worms and bacterial DNA detected by PCR with Wolbachia specific primers (ftsZ gene). Microinjected worms were cultured in vitro, and 81% survived for 10 days. Implantation of microinjected worms into Meriones unguiculatus, the rodent host of A. viteae resulted in 38% survival. The DNA of the microinjected worms recovered from jirds 8 weeks after implantation contained Wolbachia DNA as shown by PCR, suggesting that Wolbachia of L. sigmodontis can be horizontally transmitted to A. viteae.

Protective immunity induced by irradiated third-stage larvae of the filaria Acanthocheilonema viteae is directed against challenge third-stage larvae before molting.

Jirds (Meriones unguiculatus) were vaccinated with irradiated L3 third-stage larvae (L3) of Acanthocheilonema viteae, and the time required for killing of the challenge L3 was determined. The number of parasites recovered from vaccinated jirds was reduced to about 10% of the control values on the second day after challenge infection and later on. Histological studies revealed an eosinophil-rich infiltrate containing macrophages, neutrophils, and mast cells in the vicinity of the L3 on day 2 after challenge and destruction of the worms by day 4 after challenge. Ultrastructural studies confirmed these data and showed that eosinophils, macrophages, and mast cells were close to the L3 on day 2 after challenge. Flattening of the eosinophils onto the surface of the worms, degranulation of electron-dense material, and rupture of the L3 surface was observed on day 4 after challenge, followed by invasion of the inner of the worms by phagocytic cells. These data show that immune attack against the challenge L3 in vaccinated jirds is initiated between the first and the second day after challenge and that killing occurs around the fourth day after challenge, before the worms undergo their first molt.

Identification of chitinase as the immunodominant filarial antigen recognized by sera of vaccinated rodents.

Acanthocheilonema viteae is a parasitic nematode of rodents. We identified the chitinase of A. viteae infective stage larvae (L3) as the main target of the humoral immune response of jirds, which were protected against challenge infection after vaccination with irradiation attenuated L3. The cDNA of the L3 chitinase has been sequenced, and the deduced amino acid sequence shows significant homologies to chitinases of Brugia malayi microfilariae, insects, yeast, bacteria, and Streptomyces sp. The protein has been characterized by monoclonal antibodies and substrate activity gels. The chitinase of L3 may contribute to degrading the nematode cuticle during molting and thus represents a target of protective immune responses in a phase where the parasite is highly vulnerable. In addition, it has been shown that a similar enzyme exists in uterine microfilariae, which probably has a role in casting the egg shell.

Cytochemical demonstration of lectin binding-sites in the cuticle and tissues of Acanthocheilonema viteae (Filarioidea).

The lectin-gold technique was used for the ultrastructural localization of lectin binding sites on thin sections of Lowicryl K4M embedded adult females, infective larvae and SDS-2-mercaptoethanol-insoluble cuticle components of Acanthocheilonema (Dipetalonema) viteae. Helix pomatia lectin (HPL) coupled to 14 nm gold particles, was used for the demonstration of N-acetyl-D-galactosamine-containing glycoconjugates. Triticum vulgaris (wheat germ) agglutinin (WGA) coupled to 10 nm gold particles after cross-linking to BSA or ovomucoid-gold after application of unlabeled WGA, demonstrated WGA binding sites (N-acetyl-D-glucosamine). With both lectins no surface labelling of the cuticle was observed, but subcuticular layers reacted positively. HPL-gold was bound to cuticular fibers, the matrix and to the electron dense layer within the cortical zone of the cuticle of female worms. WGA-gold complexes were bound mainly to the cuticle matrix and somatic tissues. The results support the hypothesis that tissue-dwelling parasitic nematodes have reduced their surface carbohydrates perhaps as a consequence of their parasitic life.

Demonstration of anti-cuticular antibodies by immuno-electron microscopy in sera of mice immunized with cuticular extracts and isolated cuticles of adult Dipetalonema viteae (Filarioidea).

The immunogold technique was used for the ultrastructural localization of antibody-binding sites on thin sections of Lowicryl K4M embedded adult females, infective larvae and pieces of adult cuticles of Dipetalonema viteae insoluble in SDS-2-ME. The antisera used were either produced against SDS-2-ME extracts of cuticles or the insoluble pellet after SDS-2-ME extraction. With both types of antisera a labelling of epitopes on fibers was achieved in intact cuticles. In isolated cuticles the corresponding structures were absent. The same sera crossreacted with the larval and microfilarial cuticle as well as with somatic structures of all three stages. The only serum against isolated cuticle, which did not recognize cuticle fibers also did not crossreact with somatic structures. The recognition of the electron dense cortical layer insoluble in SDS-2-ME depended on the number of immunizations. A labelling of the filarial surface was never achieved. A dense labelling of the apical membrane enfoldings of the hypodermis pointed to an involvement in the synthesis of the nematode cuticle. The rough endoplasmic reticulum in the apex of the epithelial cells of the uterus, the content of the nutrient channels, and the substance between eggshell and microfilariae crossreacted with most of the antisera. This led to the conclusion that these substances are partly produced by the uterus epithelium.

The isolation and immunogenicity of the cuticle of Dipetalonema viteae (Filarioidea).

The cuticle of the filaria Dipetalonema viteae was isolated by sonication in 1% sodiumdodecylsulphate (SDS) and in a mixture of 1% SDS and 5% B-mercaptoethanol (BME). Sonication in SDS removed all internal parts and left the cuticle intact; this was verified by light- and electronmicroscopy. Sonication and incubation of the cuticle in the mixture of SDS-BME at pH 6.8 dissolved the basal and part of the median zone of the cuticle. The epicuticle and the cortical zone remained intact. The extracts were investigated using SDS-polyacrylamide gel electrophoresis; the early extracts contained a wide variety of proteins, whereas the later steps showed a consistent pattern with a smaller number of bands. Cuticles after SDS-purification, the extract of cuticular material in SDS-BME, and the cuticles insoluble in SDS-BME were used to immunize mice; the antibodies produced were visualized by an indirect fluorescent antibody test on cryostat sections of female worms. When SDS-purified cuticles were used for immunization, antibodies directed against all organs in the filariae were found. The SDS-BME extract and the insoluble cuticular pellet stimulated the production of antibodies restricted to the cuticle of adult worms and microfilariae. The purification method opens up the possibility of further isolation and characterization of antigens from the cuticle.

Neutrophil-mediated killing of Dipetalonema viteae microfilariae: simultaneous presence of IgE, IgG antibodies and complement is required.

Neutrophils from the peripheral washings of normal rats in the presence of sera obtained from rats immune to circulating microfilariae adhered to and killed the microfilariae of Dipetalonema viteae in vitro within 16-24 hr. No significant adherence or cytotoxicity was mediated by sera collected from animals with a high microfilaraemia or from normal rats. Ultrastructural studies show that neutrophils, which are bigger than microfilariae, can easily internalize the small larvae resulting in the disintegration of the parasite. Immunoadsorption and inhibition experiments showed that the adherence-promoting activity resides both in IgG and IgE classes of antibody. However, the mere participation of these two antibodies is not sufficient to effect neutrophil adherence towards microfilariae, the presence of complement is also required. Samples of fresh immune rat serum (fIRS) depleted in alternative pathway components of complement by treatment with zymosan A failed to mediate cell adherence to the parasite. fIRS inactivated for the classical pathway of complement by the chelating agent EGTA partially retains its activity in mediating cytotoxicity to microfilariae. The striking antigenic specificity of D. viteae antibodies was shown by their ability to mediate cytotoxicity only to D. viteae but not towards Brugia malayi microfilariae.

Immunocytochemical and ultrastructural studies on Dipetalonema viteae (Filarioidea).

The antigenic properties of adult male and female of Dipetalonema viteae were studied by immunocytochemistry. Using antisera of the rodents Meriones unguiculatus and Mastomys natalensis infected with D. viteae, the binding of antibodies to sections of filariae embedded in Epon was assayed by the peroxidase-antiperoxidase (PAP) technique and by electron microscopy. The optimal staining intensity was obtained with an antiserum dilution of 1:5000. Control sera were obtained from sex and age matched uninfected animals. Female D. viteae showed maximal antigen-antibody reactions within the uterus: in the inner uterus wall, in the "nutrient channels" between the maturing eggs and the differentiating microfilariae, on the eggshells, in the cuticula of microfilariae and in the spermatheca on the cell membrane of the mature spermatozoa. Male filariae showed binding of antibodies in the vesicula seminalis: in the nucleus and the nuclear membrane of primary spermatocytes and on maturing spermatids. Less pronounced antigen-antibody reactions in the cuticula, muscle and intestine were observed in both sexes. The PAP-technique offers significant improvements in comparison with other techniques, e.g., immunofluorescence, used to detect antigens on filariae: the PAP-technique has an increased sensitivity with a concomitant reduction in nonspecific background and can be used for both light and electron microscopy; moreover, PAP-treated tissues can be stored indefinitely at room temperature.

Complement-mediated leukocyte adherence to infective larvae of Dipetalonema viteae (Filarioidea): requirement for eosinophils or eosinophil products in effecting macrophage adherence.

The present study reports the existence of C-mediated adherence of eosinophils and/or macrophages to filarial infective larvae of Dipetalonema viteae. C3 molecules are present on the surface of the parasite, as shown by immunofluorescence studies. Samples of fNRS depleted of AP of complement by treatment with Zymosan A or of factor B by heating at 50 degrees C for 20 min fail to mediate cell adherence to the parasite. In contrast, fNRS inactivated for CP of complement by the chelating agent EGTA retains its activity in mediating cell adherence to the parasite. There is a significant consumption of factor B and AP of complement when infective larvae are incubated in fNHS. Consumption of C4 of the CP of complement is much lower in the same test. The adherence of macrophages cannot occur without the simultaneous presence of eosinophils, whereas eosinophils probably act alone and not in concert with macrophages. The eosinophil adherence is associated with degranulation. The damage is notably enhanced by replacing the spent eosinophil population with a newly obtained population. In the present test system, mast cells did not adhere to filarial larvae even when mast cell-rich populations were used, nor did they effect macrophage adherence when presented in association with the latter. When eosinophil-enriched cell populations containing less than 1% mast cells were used, cell adherence to filarial larvae still occurred, but the presence of 30% mast cells in such cell populations markedly increased both the rate and level of adherence. We suggest that a cell-mediated adherence and destruction dependent upon the activation of complement via AP, without a requirement for specific antibody, may represent a natural mechanism of parasite killing in a nonimmune host.

Macrophage-mediated damage to filarial worms (Dipetalonema setariosum) in vivo.

An adult female Dipetalonema setariosum (Mönnig 1926) recovered from the pleural cavity of Meriones libycus and bearing an adherent cell mass examined by means of electron microscopy. The host reaction consisted exclusively of macrophages and was associated with disruption of the cuticular membrane and invasion of the cuticle itself. There was no evidence of prior activity by other cell types. Initiation of damage appeared to be associated with the release of lysosomal enzymes by the macrophages. Within the reaction a localized breaching of the cuticle had occurred and cells had penetrated the internal tissues of the worm. It is suggested that macrophages may play a role in the elimination of effect worms from an established population.

Electron microscopic studies on microfilariae of Dipetalonema viteae and Litomosoides carinii: the occurrence of intracellular parasites after treatment with metrifonate or diethylcarbamazine.

Microfilariae of Dipetalonema viteae and Litomosoides carinii were studied by means of electron microscopy after oral treatment of their hosts with metrifonate (3 x 100 mg/kg) or diethylcarbamazine (3 x 250 mg/kg). These dosages led to the disappearance of microfilariae from the peripheral venous blood. However, in numerous organs blood capillaries or interstitial spaces contained degenerating microfilariae. In these cases the cytoplasm of the microfilarian cells was completely lysed, whereas the cuticle seemed to be unaffected, at least initially. Microfilariae of both species were also often found intracellularly especially in liver and muscle cells after application of both drugs. The intracellular microfilariae, however, had fewer or even no lesions, suggesting that they might escape the activity of the drug.

[New contribution on Dipetalonema viteae. Keeping adults alive in vitro. Scanning electron microscope study of the development of embryos and young larvae L1 and L2]. / Nouvelle contribution à l'étude de Dipetalonema viteae. Maintien en survie des adultes in vitro. Observation en microscopie électronique à balayage du développement des embryons et des jeunes larves L1 et L2.

The adult stages of Litosomosoides carinii, Dirofilaria uniformis and Dirofilaria immitis have been successfully maintained in vitro though microfilaria production by the worms continued only for a period of one to 18 days. In this paper we describe the results obtained in a series of experiments in which adult L1 and L2 stages of Dipetalonema viteae were maintained in vitro.

Dipetalonema viteae: ultrastructural study on the in vitro interaction between rat macrophages and microfilariae in the presence of IgE antibody.

The in vitro interaction between rat peritoneal macrophages and Dipetalonema viteae microfilariae in the presence of amicrofilaraemic rat immune serum was studied by transmission electron microscopy. The probable sequence of events leading to the killing of D. viteae microfilaria by macrophages is as follows. (a) Rat peritoneal macrophages in the presence of amicrofilaraemic rat immune serum adhere to the parasite surface, (b) the macrophages extend their pseudopodia around the parasite, (c) the 'lysosome-like' granules discharge their contents on to the parasite surface, (d) the lytic activity of these products begins at the parasite surface and (e) subsequent breaking of the microfilarial cuticle occurs, exposing the parasite intracellular material.

[R1 cell and musculature of Filariae; ultrastructural analysis (author's transl)]. / Cellule R1 et musculature des Filaires; analyse ultrastructurale.

Ultrastructural study of ontogenesis of a filaria, Dipetalonema (M.) dessetea, confirms Bain's (1970) hypothesis, following which the R1 cell plays a role in the elaboration of the musculature of the adult filaria. Anatomical observations related to the hypodermis and musculature of the larva (distribution of the cells, nature of their junctions, presence of intermediate cells between embryonic cells derived from R1 cell and muscle cells) show that the cells derived from R1, which form four longitudinal files, produced successively, from 5th to 13th day, about ten files of muscle cells in each submedian field. The primary musculature of the microfilaria, which corresponds to a single file of muscular cells in each field, is not ectodermic, as supposed before, but mesenchimatic. At the vicinity of R1 daughter cells, this musculature is slightly dedifferenciated (about 5 files of myofilaments instead of 10-20 in the microfilaria and the L3); then its evolution becomes identical to this of new formed muscle cells. The presence as well as the evolution of the R1 cell seem to be identical in every totally heteroxenous Phasmidian Nematodes; both suggest analogy with the imaginal discs of the holometabolic Insects.

Studies on Dipetalonema viteae (Filarioidea). 5. Ultrastructural aspects of the antibody-dependent cell-mediated destruction of microfilariae.

The antibody-dependent cell-mediated destruction of Dipetalonema viteae microfilariae was followed by electron microscopy both in vitro and within micropore chambers in vivo. There was a correlation between the degree of adherence (1% mf with adhered cells) and the degree of microfilarial damage. Polymorphonuclear leukocytes, predominantly neutrophils, seemed to be responsible for the destruction of microfilariae in vivo. An in vitro assay indicated that eosinophils also have a role as potent effectors against microfilariae. The first sign of microfilarial damage is the disintegration of cuticular layers. In a later stage of destruction, lysis of the hypodermis or even of the whole microfilarial tissues was observed.

[Dipetalonema viteae larva studied by scanning electron microscopy]. / Nouvelle contribution à l'étude de la larve de Dipetalonema viteae en microscopie électronique à balayage.

If the description in the literature is based on a study with the light microscope, in this work, with electron microscope scanning, the integumental surface on the larva of Dipetalonema viteae is studied at 1,500 to 50,000 magnifications.